Gold optimized CAR t-cells

ABSTRACT

Control Devices are disclosed including RNA destabilizing elements (RDE), RNA control devices, and destabilizing elements (DE) combined with Chimeric Antigen Receptors (CARs) or other transgenes in eukaryotic cells. Multicistronic vectors are also disclosed for use in engineering host eukaryotic cells with the CARs and transgenes under the control of the control devices. These control devices can be used to optimize expression of CARs in the eukaryotic cells so that, for example, effector function is optimized. CARs and transgene payloads can also be engineered into eukaryotic cells so that the transgene payload is expressed and delivered after stimulation of the CAR on the eukaryotic cell.

This application is continuation of U.S. application Ser. No. 16/655,175filed Oct. 16, 2019, which is a continuation of U.S. application Ser.No. 16/438,877 filed Jun. 12, 2019, now U.S. Pat. No. 10,472,637, whichis a continuation of U.S. application Ser. No. 16/151,138 filed Oct. 3,2018, now U.S. Pat. No. 10,323,249, which is a continuation of U.S.application Ser. No. 15/692,440 filed Aug. 31, 2017, now U.S. Pat. No.10,323,248, which claims priority to U.S. provisional application Ser.No. 62/382,565 filed Sep. 1, 2016, U.S. provisional application Ser. No.62/466,060 filed Mar. 2, 2017, and U.S. provisional application Ser. No.62/533,858 filed Jul. 18, 2017.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CBIO024_ST25.txt”, a creation date of Feb. 23, 2022, and asize of 8603 bytes. The Sequence Listing filed via EFS-Web is part ofthe specification and is incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

Chimeric Antigen Receptors are human engineered receptors that maydirect a T-cell to attack a target recognized by the CAR. For example,CAR T cell therapy has been shown to be effective at inducing completeresponses against acute lymphoblastic leukemia and other B-cell-relatedmalignancies and has been shown to be effective at achieving andsustaining remissions for refractory/relapsed acute lymphoblasticleukemia (Maude et al., NEJM, 371:1507, 2014). However, dangerous sideeffects related to cytokine release syndrome (CRS), tumor lysis syndrome(TLS), B-cell aplasia and on-tumor, off-target toxicities have been seenin some patients.

There are currently two extant strategies to control CAR technology. Thefirst is an inducible “kill switch.” In this approach, one or more“suicide” genes that initiate apoptotic pathways are incorporated intothe CAR construct (Budde et al. PLoS1, 2013doi:10.1371/journal.pone.0082742). Activation of these suicide genes isinitiated by the addition of AP1903 (also known as rimiducid), alipid-permeable tacrolimus analog that initiates homodimerization of thehuman protein FKBP12 (Fv), to which the apoptosis-inducing proteins acetranslationally fused. In the ideal scenario, these kill switchesendeavor to sacrifice the long-term surveillance benefit of CARtechnology to safeguard against toxicity. However, in vivo, thesesuicide switches are not likely to realize this goal, as they areoperating against powerful selection pressures for CAR T-cells that donot respond to AP1903, a situation worsened by the inimical error-proneretroviral copying associated with the insertion of stable transgenesinto patient T-cells. In this scenario, non-responsive CAR T-cell cloneswill continue to proliferate and kill target cells in anantigen-dependent manner. Thus, kill switch technology is unlikely toprovide an adequate safeguard against toxicity.

The second CAR regulatory approach is transient CAR expression, whichcan be achieved in several ways. In one approach, T-cells are harvestedfrom unrelated donors, the HLA genes are deleted by genome-editingtechnology and CAR-encoding transgenes are inserted into the genome ofthese cells. Upon adoptive transfer, these CAR T-cells will berecognized by the recipient's immune system as being foreign anddestroyed, thus the CAR exposure in this system is transient. In anothertransient CAR exposure approach, mRNA of a CAR-encoding gene isintroduced into harvested patient T-cells (Beatty, G L 2014. CancerImmunology Research 2 (2): 112-20. doi:10.1158/2326-6066.CIR-13-0170).As mRNA has a short half-life and is not replicated in the cell orstably maintained, there is no permanent alteration of theCAR-expressing T-cell, thus the CAR expression and activity will be fora short period of time. However, as with the kill-switch approach, thesetransient CAR exposure approaches sacrifice the surveillance benefit ofCARs. Additionally, with these transient systems acute toxicity can bedifficult to control.

SUMMARY OF THE INVENTION

In an aspect, the description discloses a eukaryotic cell with a CAR,T-cell receptor, or other targeting polypeptide and a transgene underthe control of an RNA Destabilizing Element (RDE). The RDE may controlmultiple transgenes or multiple RDEs may control multiple transgenes.The multiple transgenes may be arranged serially and/or as a concatemerand/or in other arrangements. Multiple RDEs may be used to regulate atransgene, and these multiple RDEs can be organized as a concatemer,interspersed within a region of the transcript, or located in differentparts of the transcript. Multiple transgenes can be regulated by an RDEor a combination of RDEs. The RDEs can be localized in the 3′-UTR, the5′-UTR and/or an intron. In an aspect, the RDE can be engineered toincrease or decrease the binding affinity of RNA binding protein(s) thatinteract with the RDE. Altering the affinity of the RNA binding proteincan change the timing and response of transgene expression as regulatedby the RNA binding protein. In an aspect, the RNA binding proteinbinding at the RDE is altered by the metabolic state of the cell andchanging the binding affinity of the RDE for the RNA binding proteinalters the response to and/or timing of transgene expression with themetabolic state of the cell. In an aspect, the RNA binding proteinbinding at the RDE is altered by the redox state of the cell andchanging the binding affinity of the RDE for the RNA binding proteinalters the response to and/or timing of transgene expression with theredox state of the cell.

In an aspect, the CAR, T-cell receptor, B-cell receptor, innate immunityreceptor, or other targeting receptor or targeting polypeptiderecognizes an antigen at the target site (e.g., tumor cell or otherdiseased tissue/cell) and this activates the cell. The transgene can beanother CAR that recognizes a second antigen at the target site andactivation of the cell by the first CAR, T-cell receptor or othertargeting polypeptide induces the second CAR allowing the eukaryoticcell to recognize the target site by a second antigen. In an aspect, theeukaryotic cell has a first CAR that recognizes an antigen at a targetsite and this activates a transgene (through an RDE) that encodes apolypeptide that directly or indirectly reduces the activation state ofthe cell. For example, the transgene may encode a second CAR thatrecognizes an antigen on healthy tissue so that when the first CARreacts with antigen at a nontarget cell, the eukaryotic cell will bede-activated by the second CAR interaction with the healthy cell antigen(that is not present or is present in reduced amounts at the targetsite).

In some aspects, the eukaryotic cell is an immune cell, e.g., a T-cell,a natural killer cell, a B-cell, a macrophage, a dendritic cell, orother antigen presenting cell. In these aspects, activation of the cellby the CAR or changing the metabolic state of the immune cell in otherways can induce expression of the transgene through the RDE. The RDEthat controls the transgene can have microRNA binding sites and can beengineered to remove one or more of these microRNA binding sites. TheRDE can be bound by the Hu Protein R (HuR). Without wishing to be boundby theory it is expected that HuR can bind to some RDEs, and act tostabilize the mRNA, leading to enhanced translation. Some RDEs can betied to the glycolytic state of the eukaryotic cell through the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH), other dehydrogenases,other oxidoreductases, or other glycolytic enzymes that can bind to anRDE when the eukaryotic cell is not activated (low glycolytic activity),quiescent, or at rest. When GAPDH or the other enzymes bind to the RDEthis can reduce half-life of the RNA with the RDE. In this aspect, CARactivation of the eukaryotic cell (e.g., T-lymphocyte) can induceglycolysis in the cell which reduces GAPDH binding of the RNA, increaseshalf-life of the RNA, which produces increased expression of thetransgene encoded in the RNA and controlled by the RDE. Without wishingto be bound by theory, as GAPDH vacates the RDE, HuR or other RDEbinding proteins may subsequently bind either the same RDE, or apreviously inaccessible RDE (sterically hindered by presence of GAPDH),further stabilizing the mRNA, increasing half-life of the mRNA, andproducing further increased expression of the transgene encoded by theRNA and controlled by said RDE. Thus, CAR activation can induceexpression of the transgene. In other aspects, other activation of theimmune cell can cause GAPDH to engage in glycolysis and so induceexpression of the transgene under the control of the RDE.

Expression from the transcript with the RDE(s) can respond to themetabolic state of the cell. For example, the RDE can be bound bymetabolic or glycolytic enzymes which couples expression of thetransgene to the activation state of the cell through these metabolic orglycolytic enzymes. Some metabolic or glycolytic enzymes bind to RDEs inthe transcript and degrade or target for degradation the transcript.When those metabolic or glycolytic enzymes become active, the enzymes nolonger bind to the RDEs, the transcripts are stable for a longer periodof time, and the transcripts can be translated for this longer period oftime. Cells expressing transgenes under the control of such RDEs canalso be engineered to express a CAR that can alter the metabolic stateof the cell at desired times resulting in expression of the transgene atthe desired time. Alternatively, other stimuli can be used to alter themetabolic state of the eukaryotic cell resulting in expression of thetransgene. For example, the metabolic state of the cell can be alteredto cause transgene expression (or to inhibit expression) by stimuliincluding, for example, small molecules (e.g., PMA/ionomycin),cytokines, a TCR and costimulatory domain engagement with ligand, oxygenlevels, cellular stress, temperature, or light/radiation.

GAPDH binding to the RDE can be increased by introducing into the cell asmall molecule that inhibits glycolysis such as, for example, rapamycin,2-deoxyglucose, 3-bromopyruvic acid, iodoacetate, fluoride, oxamate,pioglitazone, dichloroacetic acid, or other metabolism inhibitors suchas, for example, dehydroepiandrosterone. Other small molecules can beused to reduce GAPDH binding to the RDE. Such small molecules may blockthe RDE binding site of GAPDH including, for example, CGP 3466B maleateor Heptelidic acid (both sold by Santa Cruz Biotechnology, Inc.),pentalenolactone, or 3-bromopyruvic acid. Other small molecules can beused to analogously inhibit other enzymes or polypeptides from bindingto RDEs. Other small molecules can be used to change the redox state ofGAPDH, leading to an altered affinity of GAPDH for the RDE.

In an aspect, activation of the immune cell induces expression of thetransgene that can encode a payload to be delivered at the target(activation) site. The transgene can encode a payload for delivery atthe site of CAR activation and/or immune cell activation. The payloadcan be a cytokine, an antibody, a reporter (e.g., for imaging), areceptor (such as a CAR), or other polypeptide that can have a desiredeffect at the target site. The payload can remain in the cell, or on thecell surface to modify the behavior of the cell. The payload can be anintracellular protein such as a kinase, phosphatase, metabolic enzyme,an epigenetic modifying enzyme, a gene editing enzyme, etc. The payloadcan be a gene regulatory RNA, such as microRNAs, antisense RNA,ribozymes, and the like, or guide RNAs for use with CRISPR systems. Thepayload can also be a membrane bound protein such as GPCR, atransporter, etc. The payload can be an imaging agent that allows atarget site to be imaged (target site has a desired amount of targetantigen bound by the CAR). The payload can be a checkpoint inhibitor,and the CAR and/or other binding protein (e.g., T-cell receptor,antibody or innate immunity receptor) can recognize a tumor associatedantigen so the eukaryotic cell preferentially delivers the checkpointinhibitor at a tumor. The payload can be a cytotoxic compound including,for example, a granzyme, an apoptosis inducer, a cytotoxic smallmolecule, or complement. In some aspects, expression of the CAR is underthe control of an inducible promoter, an RNA control device, a DE, aSide-CAR, and/or an RDE. The amount of CAR on the surface of the cellcan allow the eukaryotic cell to be preferentially activated at thetumor site and not at normal tissue because the tumor displays higheramounts of target antigen (e.g., the amount of CAR can be adjusted toincrease avidity at the tumor site versus healthy tissue). In addition,this regulatory control of CAR expression provides another level ofcontrol to the eukaryotic cell and its delivery of payload. In anaspect, the payload can remain in the cell or on the cell surface(rather than secreted to the target), to modify the behavior of thecell.

In some aspects, the expression of CAR, DE-CAR and/or Side-CARpolypeptide is controlled, at least in part, by an RDE that interactswith a glycolytic enzyme with RDE binding activity, e.g., GAPDH. Theglycolytic enzyme can bind to the RDE and reduce production of the CAR,DE-CAR, Side-CAR polypeptide, and/or other transgene product. Thisreduction in polypeptide production can occur because of an inhibitionof translation and/or an increase in the rate of mRNA degradation (RDEbinding can shorten the half-life of the mRNA). Some RDE bindingproteins may reduce translation and enhance degradation of RNA to reducethe level of polypeptide made. The RDE can be an AU rich element fromthe 3′ UTR of a transcript (e.g., a transcript encoding IL-2 or IFN-γ),or can be a modified 3′ UTR that has been engineered to remove one ormore microRNA sites (e.g., modified 3′-UTRs of IL-2 or IFN-γ). In anaspect, the expression of the transgene, CAR, DE-CAR and/or Side-CARpolypeptide under the control of an RDE bound by a glycolytic enzyme(s),e.g., GAPDH, is increased by increasing the activity of the enzyme(s) inprosecuting glycolysis. The activity of enzymes in glycolysis can beincreased by providing the cell with increased glucose in the cellmedium, increasing triose isomerase activity in the cell, or providingthe cell with a compound that increases glycolysis in the cell, e.g.,tamoxifen or glucose. The RDE can bind to Hu Protein R (HuR). Withoutwishing to be bound by theory it is expected that HuR binds to someAU-rich RDEs and U-rich RDEs, and can act to stabilize the mRNA, leadingto enhanced translation. Thus, cell conditions that result in increasedHuR expression can increase expression of transgenes with appropriateAU-rich elements and/or U-rich elements, and conditions that reduce HuRexpression can decrease expression of these transgenes. HuR interactionwith the 3′ UTR of the transgene (or native genes) can also be alteredby expressing a recombinant transcript containing HuR binding sites.Expression of these transcripts will reduce the amount of HuR availableto bind to the transgene transcript or native HuR regulated transcriptsand reduce the half-lives of these transcripts resulting in decreasedexpression.

In an aspect, bicistronic (or multicistronic) vectors are used tointroduce two or more transgene-RDE constructs. These bicistronicconstructs can be derived from lenti virus. The two transgenes may beexpressed in opposite directions on opposite strands from controlregions located in between the nucleic acids encoding two of thetransgenes. When more than two transgenes are placed in the construct(multicistronic construct) the third (and additional) transgene may beplaced in series with one or both of the transgenes expressed inopposite directions. These additional transgenes may be expressed fromthe same control region or may have separate control regions. Onetransgene may encode a CAR and the other transgene(s) may encode payloadto be delivered when the CAR is activated. The nucleic acid encoding thepayload in the multicistronic or bicistronic construct can be controlledby an RDE that responds to the glycolytic or energy state of the cell.The transgene encoding the CAR can be operably linked to a controlregion that has a high level of transcription activity, a low level oftranscription, and/or is an inducible promoter, and the transgeneencoding the payload can be operably linked to a control region that hasa lower level of transcription activity, a higher level oftranscription, and/or is inducible. The CAR can be operably linked to acontrol region that has a higher level of transcription (and/or isinducible) and the transgene encoding the payload can be operably linkedto a control region that has a lower level of transcription (and/or isinducible). The CAR can also be operably linked to a control region thathas a lower level of transcription (and/or is inducible) and thetransgene encoding the payload can be operably linked to a controlregion that has a higher level of transcription (and/or is inducible).

In an aspect, nucleic acids can be used to boost the response of immunecells upon stimulation of the immune cell. For example, the immune cellcan produce higher amounts of immune polypeptides (greater C_(max)) withfaster kinetics of production. The immune polypeptides can include, forexample, cytokines, perforins, granzymes, apoptosis inducingpolypeptides, etc. The nucleic acids that boost the immune response cancomprise control regions operably linked to nucleic acids encoding RDEsfor selected RDE binding proteins, so that upon expression of thenucleic acid into RNA the RDEs in the RNA bind the RDE binding proteinsthat repress expression of a polypeptide, for example, cytokines,perforins, granzymes, and other immune polypeptides. The expression ofthe RNAs with the RDEs can poise the eukaryotic cell for expression ofpolypeptide controlled by RDEs. For example, the expression of RNAs withthe RDEs may be done in immune cells to poise the cell for expression ofimmune polypeptides upon stimulation of the immune cell.

Certain RDEs can be associated with certain disease states in a subject.Some disease associated RDEs can be found by comparing the RDEs in thetranscripts from normal cells (tissue) to RDEs in the transcripts fromdiseased or aberrant cells (tissue). RDEs and their corresponding RNAbinding proteins from a normal (healthy) cell(s) can be compared tothose in a diseased or aberrant cell(s) by trapping the RDE with its RNAbinding proteins using methods described in Castello et al., Molc. Cell63:696-710 (2016), which is incorporated by reference in its entiretyfor all purposes. RDEs that have aberrant interactions with RNA bindingproteins can be linked to a disease state and sequencing of RDEs in thegenes or transcripts from an individual may show the susceptibility todisease and/or the disease state of the subject.

RDE control of a CAR, transgene payload, and/or transgene can becontrolled through an RDE that is responsive to the metabolic state ofthe eukaryotic cell. For example, the RDE can be bound by a glycolyticenzyme or other metabolic enzyme and expression of the CAR, transgenepayload, and/or transgene can be inhibited by changing the metabolicstate of the eukaryotic cell. The RDE could be bound by GAPDH and byturning off glycolysis in the cell (e.g., using an inhibitor ofglycolysis) the expression of the CAR, transgene payload, and/ortransgene can be inhibited. This inhibition of expression can be used toreduce adverse events caused by expression of the CAR, transgenepayload, and/or transgene.

In an aspect, the CAR, DE-CAR, Side-CAR polypeptides, and/or otherreceptor can be directed against antigens found on acute myeloidleukemia (AML) cells including, for example, CD 33, CD 34, CD 38, CD 44,CD 45, CD 45RA, CD 47, CD 64, CD 66, CD 123, CD 133, CD 157, CLL-1,CXCR4, LeY, PR1, RHAMM (CD 168), TIM-3, and/or WT1. The monoclonalantibody 293C3-SDIE can be used as the extracellular element for theCAR, DE-CAR and/or Side-CAR polypeptides. (Rothfelder et al., 2015, atash.confex.com/ash/2015/webprogram/Paper81121.html, which isincorporated by reference in its entirety for all purposes) Otherantigens for AML are known in the art and may be the target of the CAR,DE-CAR, Side-CAR, and/or other receptor. In an aspect, the CAR, DE-CAR,Side-CAR polypeptides, and/or other receptor can be directed againstantigens found on diffuse large cell B-cell lymphoma (DLBCL) cellsincluding, for example, CD19, CD20, CD22, CD79a, CD5, CD10, and CD43.Other antigens for DLBCL are known in the art and may be the target ofthe CAR, DE-CAR, Side-CAR, and/or other receptor.

In an aspect, the desired amount of CAR expression may consider thetarget cell concentration, density of target antigen on target cells,the binding affinity (K_(d)) of the extracellular element (antigenbinding element) for the target antigen, the concentration of eukaryoticcells with CARs. These parameters and other parameters may be used toarrive at a desired density of CARs on the eukaryotic cell which willdefine the desired level of CAR expression. The desired amount of CARexpression can also consider the amount of inhibitory receptors (IR)expressed on the eukaryotic cell, and the amount of inhibitory receptorligand (IRL) expressed on target (and other) cells. The followingequations can be used, at least in part, to arrive at a desired amountof CAR polypeptide:

$\begin{matrix}\begin{matrix}{{{Cell}\mspace{14mu}{Activity}} = {{{\left\lbrack {{target}\mspace{14mu}{cell}} \right\rbrack\left\lbrack {{target}\mspace{14mu}{antigen}\mspace{14mu}{density}} \right\rbrack}\left\lbrack K_{d} \right\rbrack}\quad}} \\\left\lbrack {{eukaryotic}\mspace{14mu}{cells}} \right\rbrack\end{matrix} & I \\\frac{\begin{matrix}{{{Cell}\mspace{14mu}{Activity}} = {{\left\lbrack {{target}\mspace{14mu}{cell}} \right\rbrack\left\lbrack {{target}\mspace{14mu}{antigen}\mspace{14mu}{density}} \right\rbrack}\left\lbrack K_{d} \right\rbrack}} \\\left\lbrack {{eukaryotic}\mspace{14mu}{cells}} \right\rbrack\end{matrix}}{\lbrack{IR}\rbrack\lbrack{IRL}\rbrack} & {II}\end{matrix}$

The desired amount of CAR expression can produce a desired number ofCARs on the surface of the eukaryotic cell. The desired amount of CARexpression can produce 2-100,000 CARs (or DE-CARs or Side-CARs) on thesurface of the eukaryotic cell. The eukaryotic cell can be aT-lymphocyte and the number of CARs (or DE-CARs or Side-CARs) on thesurface of the T-lymphocyte can be 2-100,000. The CAR, DE-CAR, and/orSide-CAR can bind to target ligand with an affinity in the micromolar(μM) range and the desired number of CARs, DE-CARs, and/or Side-CARs onthe surface of the T-lymphocyte or natural killer cell can be100-500,000. The CAR, DE-CAR, and/or Side-CAR can bind to target ligandwith an affinity in the nanomolar (nM) range and the desired number ofCARs, DE-CARs, and/or Side-CARs on the surface of the T-lymphocyte ornatural killer cell can be 2-100,000.

A nucleic acid construct encoding a transcript with selected RDEs can beexpressed in an immune cell, for example, a T-lymphocyte. Therecombinant transcript with the selected RDEs can bind to and depletethe levels of RDE binding proteins in the T-lymphocyte so thattranscripts encoding polypeptides regulated by the depleted RDE bindingproteins are expressed at different threshold points of activation forother cellular signals. The use of the RDE constructs can increase thekinetics of expression and/or the Cmax of expression of the polypeptideswhose expression is controlled by the RDE.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic diagram of a chimeric antigen receptor—RNAcontrol device (Smart CAR).

FIG. 2 provides a schematic diagram of a chimeric antigenreceptor—Destabilizing Element (DE-CAR).

FIG. 3 provides a schematic diagram of a chimeric antigenreceptor—Destabilizing Element—RNA control device (Smart-DE-CAR).

FIG. 4 provides a graph showing effector function (E), activationsignaling (N_(A)) and inhibitory signaling (N_(I)) over time for anumber of CAR receptors.

FIG. 5 provides a diagram showing effector function, T-cell capabilityand T-cell exhaustion (or dysfunction) for CD8+ T-cells receivingcontinuous antigen stimulation over time.

FIG. 6 provides a graph of cell killing activity for anti-CD19 CAR, CD8+T-cells over time in the presence of different concentrations oftheophylline (ligand for the RNA control device).

FIG. 7 provides a graph for a CAR T-lymphocyte showing time versuseffector function (E), number of target cells (nT), number of CARreceptors (nR), and number of CAR receptor-target cell interactions(nRT).

FIG. 8 shows a diagram for optimal CAR activity where the threevariables are CAR copy number, target epitope copy number and CARbinding affinity.

FIG. 9 depicts a new tetracycline RNA control device (SEQ ID NO: 1).

FIG. 10 depicts an alternative new tetracycline RNA control device (SEQID NO: 2).

FIG. 11 depicts an aptamer that binds 6R-folinic acid (SEQ ID NO: 3).

FIG. 12 depicts an alternative aptamer that binds 6R-folinic acid (SEQID NO: 4).

FIG. 13 depicts a new 6R folinic acid RNA control device (SEQ ID NO: 5).

FIG. 14 depicts an alternative 6R folinic acid RNA control device (SEQID NO: 6).

FIG. 15 depicts a still further alternative 6R folinic acid RNA controldevice (SEQ ID NO: 7).

FIG. 16 shows a graph for the bioluminescence from T-cells withluciferase controlled by an RDE following activation of the T-cell byRaji target cells (activate CAR) or by CD3/CD28 beads (activate TCR) ascompared to bioluminescence of T-cells at resting.

FIG. 17 shows a graph for bioluminescence from T-cells with luciferasecontrolled by the RDEs Gold1, Gold2, or Gold3 following activation ofthe T-cell by Raji target cells (activate CAR) as compared tobioluminescence of T-cells at resting.

FIG. 18 shows a graph for the IL-12 expression from T-cells with IL-12expression controlled by an RDE following activation of the T-cell byRaji target cells (activate CAR) as compared to IL-12 expression ofT-cells at resting.

DETAILED DESCRIPTION OF THE INVENTION

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimscan be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.Numerical limitations given with respect to concentrations or levels ofa substance are intended to be approximate, unless the context clearlydictates otherwise. Thus, where a concentration is indicated to be (forexample) 10 it is intended that the concentration be understood to be atleast approximately or about 10 μg.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

As used herein, an “actuator element” is defined to be a domain thatencodes the system control function of the RNA control device. Theactuator domain can optionally encode the gene-regulatory function.

As used herein, an “antibody” is defined to be a protein functionallydefined as a ligand-binding protein and structurally defined ascomprising an amino acid sequence that is recognized by one of skill asbeing derived from the variable region of an immunoglobulin. An antibodycan consist of one or more polypeptides substantially encoded byimmunoglobulin genes, fragments of immunoglobulin genes, hybridimmunoglobulin genes (made by combining the genetic information fromdifferent animals), or synthetic immunoglobulin genes. The recognized,native, immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes and multiple D-segments andJ-segments. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. Antibodies exist as intact immunoglobulins, as a number ofwell characterized fragments produced by digestion with variouspeptidases, or as a variety of fragments made by recombinant DNAtechnology. Antibodies can derive from many different species (e.g.,rabbit, sheep, camel, human, or rodent, such as mouse or rat), or can besynthetic. Antibodies can be chimeric, humanized, or humaneered.Antibodies can be monoclonal or polyclonal, multiple or single chained,fragments or intact immunoglobulins.

As used herein, an “antibody fragment” is defined to be at least oneportion of an intact antibody, or recombinant variants thereof, andrefers to the antigen binding domain, e.g., an antigenic determiningvariable region of an intact antibody, that is sufficient to conferrecognition and specific binding of the antibody fragment to a target,such as an antigen. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, and Fv fragments, scFv antibodyfragments, linear antibodies, single domain antibodies such as sdAb(either V_(L) or V_(H)), camelid VHH domains, and multi-specificantibodies formed from antibody fragments. The term “scFv” is defined tobe a fusion protein comprising at least one antibody fragment comprisinga variable region of a light chain and at least one antibody fragmentcomprising a variable region of a heavy chain, wherein the light andheavy chain variable regions are contiguously linked via a shortflexible polypeptide linker, and capable of being expressed as a singlechain polypeptide, and wherein the scFv retains the specificity of theintact antibody from which it is derived. Unless specified, as usedherein an scFv may have the V_(L) and V_(H) variable regions in eitherorder, e.g., with respect to the N-terminal and C-terminal ends of thepolypeptide, the scFv may comprise V_(L)-linker-V_(H) or may compriseV_(H)-linker-V_(L).

As used herein, an “antigen” is defined to be a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. The skilled artisan will understand that anymacromolecule, including, but not limited to, virtually all proteins orpeptides, including glycosylated polypeptides, phosphorylatedpolypeptides, and other post-translation modified polypeptides includingpolypeptides modified with lipids, can serve as an antigen. Furthermore,antigens can be derived from recombinant or genomic DNA. A skilledartisan will understand that any DNA, which comprises a nucleotidesequences or a partial nucleotide sequence encoding a protein thatelicits an immune response therefore encodes an “antigen” as that termis used herein. Furthermore, one skilled in the art will understand thatan antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be synthesized or can be derived from a biologicalsample, or can be a macromolecule besides a polypeptide. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a fluid with other biological components.

As used herein, the terms “Chimeric Antigen Receptor” and the term “CAR”are used interchangeably. As used herein, a “CAR” is defined to be afusion protein comprising antigen recognition moieties andcell-activation elements.

As used herein, a “CAR T-cell” or “CAR T-lymphocyte” are usedinterchangeably, and are defined to be a T-cell containing thecapability of producing CAR polypeptide, regardless of actual expressionlevel. For example a cell that is capable of expressing a CAR is aT-cell containing nucleic acid sequences for the expression of the CARin the cell.

As used herein, a “costimulatory element” or “costimulatory signalingdomain” or “costimulatory polypeptide” are defined to be theintracellular portion of a costimulatory polypeptide. A costimulatorypolypeptide can be represented in the following protein families: TNFreceptor proteins, Immunoglobulin-like proteins, cytokine receptors,integrins, signaling lymphocytic activation molecules (SLAM proteins),and activating natural killer cell receptors. Examples of suchpolypeptides include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40,ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1),CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, MyD88, and thelike.

As used herein, a “Cmax” is defined to mean the maximum concentration ofa polypeptide produced by a cell after the cell is stimulated oractivated to produce the polypeptide.

As used herein, a “cytokine C_(max)” is defined to mean the maximumconcentration of cytokine produced by an immune cell after stimulationor activation to produce the cytokine.

As used herein, a “cytotoxic polypeptide C_(max)” is defined to mean themaximum concentration of cytotoxic polypeptide produced by an immunecell after stimulation or activation to produce the cytotoxicpolypeptide.

As used herein, a “destabilizing element” or a “DE” or a “Degron” areused interchangeably, and are defined to be a polypeptide sequence thatis inducibly resistant or susceptible to degradation in the cellularcontext by the addition or subtraction of a ligand, and which confersthis stability modulation to a co-translated polypeptide to which it isfused in cis.

As used herein, an “effective amount” or “therapeutically effectiveamount” are used interchangeably, and defined to be an amount of acompound, formulation, material, or composition, as described hereineffective to achieve a particular biological result.

As used herein, an “epitope” is defined to be the portion of an antigencapable of eliciting an immune response, or the portion of an antigenthat binds to an antibody. Epitopes can be a protein sequence orsubsequence that is recognized by an antibody.

As used herein, an “expression vector” and an “expression construct” areused interchangeably, and are both defined to be a plasmid, virus, orother nucleic acid designed for protein expression in a cell. The vectoror construct is used to introduce a gene into a host cell whereby thevector will interact with polymerases in the cell to express the proteinencoded in the vector/construct. The expression vector and/or expressionconstruct may exist in the cell extrachromosomally or integrated intothe chromosome. When integrated into the chromosome the nucleic acidscomprising the expression vector or expression construct will be anexpression vector or expression construct.

As used herein, an “extracellular element” is defined as the antigenbinding or recognition element of a Chimeric Antigen Receptor.

As used herein, a “hematopoietic cell” is defined to be a cell thatarises from a hematopoietic stem cell. This includes but is not limitedto myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes,erythrocytes, mast cells, myeloblasts, basophils, neutrophils,eosinophils, macrophages, thrombocytes, monocytes, natural killer cells,T lymphocytes, B lymphocytes and plasma cells.

As used herein, “heterologous” is defined to mean the nucleic acidand/or polypeptide are not homologous to the host cell. For example, aconstruct is heterologous to a host cell if it contains some homologoussequences arranged in a manner not found in the host cell and/or theconstruct contains some heterologous sequences not found in the hostcell.

As used herein, an “intracellular element” is defined as the portion ofa Chimeric Antigen Receptor that resides on the cytoplasmic side of theeukaryotic cell's cytoplasmic membrane, and transmits a signal into theeukaryotic cell. The “intracellular signaling element” is that portionof the intracellular element which transduces the effector functionsignal which directs the eukaryotic cell to perform a specializedfunction.

As used herein, “RNA destabilizing element” or “RDE” are usedinterchangeably and both are defined as a nucleic acid sequence in anRNA that is bound by proteins and which protein binding changes thestability and/or translation of the RNA. Examples of RDEs include ClassI AU rich elements (ARE), Class II ARE, Class III ARE, U rich elements,GU rich elements, and stem-loop destabilizing elements (SLDE). Withoutwishing to be bound by theory, RDE's may also bind RNA stabilizingpolypeptides like HuR.

As used herein, an “RNase III substrate” is defined to be an RNAsequence motif that is recognized and cleaved by an endoribonuclease ofthe RNase III family.

As used herein, an “RNAi substrate” is defined to be an RNA sequencethat is bound and/or cleaved by a short interfering RNA (siRNA)complexed to an effector endonuclease of the Argonaute family.

As used herein, a “single chain antibody” (scFv) is defined as animmunoglobulin molecule with function in antigen-binding activities. Anantibody in scFv (single chain fragment variable) format consists ofvariable regions of heavy (V_(H)) and light (V_(L)) chains, which arejoined together by a flexible peptide linker.

As used herein, a “T-lymphocyte” or T-cell” is defined to be ahematopoietic cell that normally develops in the thymus. T-lymphocytesor T-cells include, but are not limited to, natural killer T cells,regulatory T cells, helper T cells, cytotoxic T cells, memory T cells,gamma delta T cells and mucosal invariant T cells.

As used herein, “transfected” or “transformed” or “transduced” aredefined to be a process by which exogenous nucleic acid is transferredor introduced into a host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

As used herein, a “transmembrane element” is defined as the elementbetween the extracellular element and the intracellular element. Aportion of the transmembrane element exists within the cell membrane.

Destabilizing Elements

Destabilizing elements (DE) are stability-affecting polypeptides capableof interacting with a small-molecule ligand, the presence, absence, oramount of which ligand is used to modulate the stability of theDE-polypeptide of interest. The polypeptide of interest can be animmunomodulatory polypeptide. The polypeptide of interest can also be aCAR. Binding of ligand by a DE-CAR can reduce the degradation rate ofthe DE-CAR polypeptide in the eukaryotic cell. Binding of ligand by theDE-CAR can also increase the degradation rate of the DE-CAR in theeukaryotic cell.

Exemplary destabilizing elements or DEs are described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, both of which areincorporated by reference in their entirety for all purposes. Forexample, U.S. Ser. No. 15/070,352 describes DEs derived from variants ofthe FKBP protein, variants of the DHFR protein, variant estrogenreceptor binding domain (ERBD), and variant phototropin 1 of Avenasativa (AsLOV2). Other examples of variant FKBP nucleic acids andpolypeptides are described in US published patent application20120178168 A1 published on Jul. 12, 2012, which is hereby incorporatedby reference in its entirety for all purposes. Other examples of variantDHFR nucleic acids and polypeptides are described in US published patentapplication 20120178168 A1 published on Jul. 12, 2012, which is herebyincorporated by reference in its entirety for all purposes. Otherexamples of variant ERBD nucleic acids, polypeptides, and ligands aredescribed in published US patent application 20140255361, which ishereby incorporated by reference in its entirety for all purposes. Otherexamples of variant AsLOV2 DEs are described in Bonger et al., ACS Chem.Biol. 2014, vol. 9, pp. 111-115, and Usherenko et al., BMC SystemsBiology 2014, vol. 8, pp. 128-143, which are incorporated by referencein their entirety for all purposes.

Other DEs can be derived from other ligand binding polypeptides asdescribed in U.S. patent application Ser. No. 15/070,352 filed on Mar.15, 2016, and U.S. patent application Ser. No. 15/369,132 filed Dec. 5,2016, both of which are incorporated by reference in their entirety forall purposes.

Other ligand binding polypeptides from which variants can be made foruse as DEs, include for example, enzymes, antibodies or antibodyfragments or antibody fragments engineered by recombinant DNA methodswith the variable domain, ligand binding receptors, or other proteins.Examples of enzymes include bromodomain-containing proteins, FKBPvariants, or prokaryotic DHFR variants. Examples of receptor elementsuseful in making DEs include: variant ERBD, or other receptors that haveligands which are nontoxic to mammals, especially humans.

The ligand(s) for the DE can be selected for optimization of certainattributes for therapeutic attractiveness, for example, as described inU.S. patent application Ser. No. 15/070,352 filed on Mar. 15, 2016, andU.S. patent application Ser. No. 15/369,132 filed Dec. 5, 2016, both ofwhich are incorporated by reference in their entirety for all purposes.

RNA Control Devices

The Ribonucleic acid (RNA) control devices disclosed herein can exhibittunable regulation of gene expression, design modularity, and targetspecificity. The RNA control devices can act to rewire information flowthrough cellular networks and reprogram cellular behavior in response tochanges in the cellular environment. In regulating polypeptideexpression, the RNA control devices can serve as synthetic cellularsensors to monitor temporal and spatial fluctuations in the levels ofdiverse input molecules. RNA control devices represent powerful toolsfor constructing ligand-controlled gene regulatory systems tailored tomodulate the expression of CAR, DE-CAR, and/or Side-CAR polypeptides ofthe invention in response to specific effector molecules enabling RNAregulation of target CAR, DE-CAR, and/or Side-CAR constructs in variousliving systems.

The RNA control devices disclosed herein comprise a regulatory elementand a sensor element. The RNA control devices disclosed herein cancomprise a single element with both a regulatory and sensory function.The RNA control devices disclosed herein can comprise a regulatoryfunction and a sensory function. The RNA control devices disclosedherein can comprise a regulatory element, a sensor element, and aninformation transmission element (ITE) that functionally couples theregulatory element and the sensor element. The ITE can be based on, forexample, a strand-displacement mechanism, an electrostatic interaction,a conformation change, or a steric effect. The sensing function of theRNA control device leads to a structural change in the RNA controldevice, leading to altered activity of the acting function. Somemechanisms whereby these structural changes can occur include stericeffects, hydrophobicity driven effects (log p), electrostatically driveneffects, nucleotide modification effects (such as methylation,pseudouradination, etc.), secondary ligand interaction effects and othereffects. A strand-displacement mechanism can use competitive binding oftwo nucleic acid sequences (e.g., the competing strand and the RNAcontrol device strand) to a general transmission region of the RNAcontrol device (e.g., the base stem of the aptamer) to result indisruption or restoration of the regulatory element in response toligand binding to the sensor element.

The RNA control device can comprise a sensor element and a regulatoryelement. The sensor element can be an RNA aptamer. The RNA controldevice can have more than one sensor element. In some aspects, theregulatory element can be a ribozyme. The ribozyme can be a hammerheadribozyme. The ribozyme can also be a hairpin ribozyme, or a hepatitisdelta virus (HDV) ribozyme, or a Varkud Satellite (VS) ribozyme, a glmSribozyme, and/or other ribozymes known in the art.

The RNA control device or devices can be embedded within a DNA sequence.The RNA control device can be encoded for in messenger RNA. Multiple RNAcontrol devices can be encoded in cis with a transgene-encoding mRNA.The multiple RNA control devices can be the same and/or a mixture ofdifferent RNA control devices repeated. The nucleic acid that is used toencode the RNA control device can be repeated. By including multiple RNAcontrol devices, sensitivity and dose response may be tailored oroptimized. The multiple RNA control devices can each be specific for adifferent ligand. This can mitigate unintentional expression due toendogenously produced ligands that interact with the sensor element.

RNA Control Devices: Sensor Elements

Exemplary sensor elements are described in U.S. patent application Ser.No. 15/070,352 filed on Mar. 15, 2016, and U.S. patent application Ser.No. 15/369,132 filed Dec. 5, 2016, both of which are incorporated byreference in their entirety for all purposes. Sensor elements can bederived from aptamers. An “aptamer” is a nucleic acid molecule, such asRNA or DNA that is capable of binding to a specific molecule with highaffinity and specificity (Ellington et al., Nature 346, 818-22 (1990);and Tuerk et al., Science 249, 505-10 (1990), which are herebyincorporated by reference in their entirety for all purposes). For areview of aptamers that recognize small molecules, see Famulok, Science9:324-9 (1999), which is hereby incorporated by reference in itsentirety for all purposes.

The binding affinity of the aptamer for its ligand must be sufficientlystrong and the structure formed by the aptamer when bound to its ligandmust be significant enough so as to switch an RNA control device of theinvention between “on” and “off” states. The association constant forthe aptamer and associated ligand is such that the ligand(s) bind to theaptamer and has the desired effect at a concentration of ligand obtainedupon administration of the ligand to a subject. For in vivo use, forexample, the association constant should be such that binding occurswell below the concentration of ligand that can be achieved in the serumor other tissue, or well below the concentration of ligand that can beachieved intracellularly since cellular membranes may not besufficiently permeable to allow the intracellular ligand concentrationto approach the level in the serum or extracellular environment. Therequired ligand concentration for in vivo use can also be below thatwhich could have undesired effects on the subject.

Ligands for RNA Control Devices

RNA control devices can be controlled via the addition of exogenousligand or synthesis (or addition) of endogenous ligands with desiredbinding properties, kinetics, bioavailability, etc., for example, asdescribed in U.S. patent application Ser. No. 15/070,352 filed on Mar.15, 2016, and U.S. patent application Ser. No. 15/369,132 filed Dec. 5,2016, both of which are incorporated by reference in their entirety forall purposes.

The ligand can be a naturally occurring, secreted metabolite. Forexample, a ligand that is uniquely produced by a tumor, or present inthe tumor microenvironment is the ligand for the sensor element andbinding of this ligand to the sensor element changes the activity of theRNA control device. Thus the control device is responsive and controlledthrough chemical signaling or proximity to a tumor.

The ligand can be selected for its pharmacodynamic or ADME behavior. Forexample ligands may be preferentially localized to specific portions ofthe human anatomy and physiology. For example certain molecules arepreferentially absorbed or metabolized in the gut, the liver, the kidneyetc. The ligand can be selected to demonstrate preferentialpharmacodynamic behavior in a particular organ. For example, it would beuseful to have a ligand that preferentially localizes to the colon for acolorectal carcinoma so that the peak concentration of the ligand is atthe required site, whereas the concentrations in the rest of the body isminimized, preventing undesired, nonspecific toxicity. The ligand can beselected to demonstrate non preferential pharmacodynamic behavior. Forexample, for disseminated tumors like hematological malignancies, itwould be useful to have non variant concentration of the ligandthroughout the body.

The ligand for the RNA control device (or DE) can be folinic acid,S-folinic acid, R-folinic acid, vitamin C (ascorbic acid), acyclovir, orthe like.

RNA Control Devices: Regulatory Elements

The regulatory element can comprise a ribozyme, or an antisense nucleicacid, or an RNAi sequence or precursor that gives rise to a siRNA ormiRNA, or a shRNA or precursor thereof, or an RNAse III substrate, or analternative splicing element, or a transcription terminator, or aribosome binding site, or an IRES, or a polyA site. Regulatory elementsuseful in the present invention are, for example, described in U.S.patent application Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S.patent application Ser. No. 15/369,132 filed Dec. 5, 2016, both of whichare incorporated by reference in their entirety for all purposes.

General approaches to constructing oligomers useful in antisensetechnology have been reviewed, for example, by van der Krol et al.(1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668, which are hereby incorporated by reference in theirentirety for all purposes. Certain miRNAs that may be used in theinvention are described in Brennecke et al., Genome Biology 4:228(2003); Kim et al., Mol. Cells. 19:1-15 (2005), which are herebyincorporated by reference in their entirety for all purposes.

The RNA control devices can have multiple regulatory elements, and/ormultiple sensor elements. The multiple sensor elements can recognize thesame or different ligands. The multiple sensor elements can havedifferent (e.g., incremental, additive or synergistic) effects on theregulatory element.

RNA Destabilizing Elements

RNA destabilizing elements (RDE) are nucleic acids that affect ormaintain the stability of an RNA molecule or the translation kinetics ofan RNA molecule. Some RDEs are bound by polypeptides which destabilize(e.g., cleave) the RNA, or prevent translation, leading to loss offunction for the RNA. Some RDE binding polypeptide stabilizes the RNAincreasing the half-life of the RNA. RDEs can be used to control theexpression of a transgene, e.g., a transgene encoding a chimeric antigenreceptors. RDEs can be used with RNA control devices, DEs, and/or SideCARs to regulate the expression of a transgene. The RDEs can also beused to control expression of transgenes encoding polypeptides otherthan a CAR. Other transgenes may encode, for example, a cytokine, anantibody, a checkpoint inhibitor, a granzyme, an apoptosis inducer,complement, a cytotoxic small molecule, other cytotoxic compounds, apolypeptide for imaging, or other polypeptide that can have a desiredeffect. The RDE can control the delivery of a transgene payload.Examples of RDEs include, for example, AU rich elements, U richelements, GU rich elements, and certain stem-loop elements. ExemplaryRDEs are described in Kovarik et al., Cytokine 89:21-26 (2017); Ray etal., Nature 499:172-177 (2013); Castello et al., Cell 149:1393-1406(2012); Vlasova et al., Molc. Cell. 29:263-270 (2008); Barreau et al.,Nucl. Acids Res. vol 33, doi:10.1093/nar/gki1012 (2006); Meisner et al.,ChemBioChem 5:1432-1447 (2004); Guhaniyogi et al., Gene 265:11-23(2001), all of which are incorporated by reference in their entirety forall purposes.

The RDE can be a Class I AU rich element (dispersed AUUUA (SEQ ID NO:8)in U rich context), a Class II AU rich element (overlapping(AUUUA)_(n)), a Class III AU rich element (U-rich stretch), a stem-loopdestabilizing element (SLDE), a cytokine 3′ UTR (e.g., INF-γ, IL-2,T-cell receptor α chain, TNFα, IL-6, IL-8, GM-CSF, G-CSF etc.), and asequence of AUUUAUUUAUUUA (SEQ ID NO: 9). Khabar, WIREs RNA 2016, doi:10.1002/wrna.1368 (2016); Palanisamy et al, J. Dent. Res. 91:651-658(2012), both of which are incorporated by reference in their entiretyfor all purposes. The RDE can also be a GU rich element comprised of oneor more of, for example, UUGUU (SEQ ID NO: 10), UGGGGAU (SEQ ID NO: 11),or GUUUG (SEQ ID NO: 12). The RDE can be a U-rich element comprised ofone or more of, for example, UUUGUUU (SEQ ID NO: 13), U (SEQ ID NO: 14),UUUAUUU (SEQ ID NO: 15), U (SEQ ID NO: 16), UUAGA (SEQ ID NO: 17), orAGUUU (SEQ ID NO: 18). In some aspects, multiple RDEs can be combined tomake a regulatory unit, for example, multiple RDEs that have the samesequence can be arranged in a concatemer or can be arranged withintervening sequence in between some or all of the RDEs. The RDEsequence can be modified to increase or decrease the affinity of an RNAbinding protein(s) for the RDE. For example, an AU rich RDE can bechanged to alter the affinity of glyceraldehyde phosphate dehydrogenase(GAPDH) to the RDE. This change in affinity can alter theGAPDH-activation threshold for expression of a transgene regulated bythe RDE to which GAPDH binds.

The RDE can be from the 3′ UTR of a gene encoding, for example, IL-1,IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, GM-CSF, G-CSF, VEG F, PGE₂, COX-2,MMP (matrix metalloproteinases), bFGF, c-myc, c-fos, betal-AR, PTH,interferon-gamma, MyoD, p21, Cyclin A, Cyclin B1, Cyclin D1, PAI-2, NOSHANOS, TNF-alpha, interferon-alpha, bcl-2, interferon-beta, c-jun,GLUT1, p53, Myogenin, NF-M, or GAP-43, lymphocyte antigen 96, SUPV3L1,SFtPA2, BLOC1S2, OR10A6, OR8D1, TRPT1,CIP29, EP400, PLE2, H3ST3A1,ZNF571, PPP1R14A, SPAG4L, OR10A6 and KIR3DL. Other RDEs are found in,for example, the 3′-UTRs from GLMN, AMY2B, AMY2A, AMY2A, AMY1A, TRIM33,TRIM33, TRIM33, CSRP1, PPP1R12B, KCNH1, Reticulon 4, MRPL30, Nav1.2,Tissue_factor_pathway_inhibitor, EEF1B2, CRYGB, ARMC9, RPL15, EAF2,MRPS22, MRPS22, COPB2, PDCD10, RE1-silencing_transcription_factor,Amphiregulin, AP1AR, TLR3, SKP2,Peptidylglycine_alpha-amidating_monooxygenase, TNFAIP8, Interleukin_9,PCDHA2, PCDHA12, Aldehyde_dehydrogenase_5_family,_member_A1, KCNQ5,COX7A2, Monocarboxylate_transporter_10, MLLT4, PHF10, PTPN12,MRNA_(guanine-N7−)-methyltransferase, WHSC1L1,Tricho-rhino-phalangeal_syndrome_Type_1, Interferon_alpha-1, ZCCHC6,Retinitis_pigmentosa_GTPase_regulator, MED14, CLCN5, DNA2L, OR52D1,NELL1, SLC22A25, SLC22A10, TRPC6, CACNA2D4, EPS8, CT2_(gene),Mitochondrial_ribosomal_protein_L42, TAOK3, NUPL1,Endothelin_receptor_type_B,Survival_of_motor_neuron_protein-interacting_protein_1, POLE2,Hepatic_lipase, TPSG1, TRAP1, RPS15A, HS3ST3A1, CROP_(gene),Apolipoprotein_H, GRB2, CEP76, VPS4B, Interleukin_28B, IZUMO1, FGF21,PPP1R15A, LIN7B, and CDC45-related_protein.

Still other RDEs can be found in, for example, the 3′UTRs of SCFD1,MAL2, KHSRP, IQCB1, CAMP_responsive_element_modulator, MFAP5, SBF2,FKBP2, PDCD10, UBE2V2, NDUFAB1, Coiled-Coil_Domain_Containing_Protein,ALG13, TPTE, Enaptin, Thymopoietin, Delta-like_1, C11orf30,Actinin_alpha_4, TMEM59, SP110, Dicer, TARDBP, IFNA17, IFNA16, IFNA14,ZMYM3, Interleukin_9,_type_I, OPN1SW, THSD1, ERGIC2, CAMK2B, WDR8, FXR1,Thymine-DNA_glycosylase, Parathyroid_hormone-related_protein, OSBPL3,Ran, GYPE, AKAP4, LOC642658, L2HGDH, AKAP1, Zinc_finger_protein_334,TC2N, FKBPL, GRB14, CXorf67, CXorf66, CEP76, Gastricsin, CEP70, CYP26A1,NAA35, Aryl_hydrocarbon_receptor_nuclear_translocator, KLC4, GPR112,LARP4, NOVA1, UBE2D3, ITGA6, GPR18, MGST_type_A,RE1-silencing_transcription_factor, ASPM, ZNF452, KIR2DS4, AHSA1, TMTC4,VSX1, P16, MRPL19, CCL20, TRPT1, Hepatic_lipase, PDLIM5, CCDC53,′CCDC55,GAPVD1, HOXB2, KCNQ5, BRCC3, GTF2IRD1, CDK5RAP3,Transcription_factor_II_B, ZEB1, IRGM, SLC39A6, RHEB, PSIP1, RPS6KA5,Urokinase_receptor, GFM1, DNAJC7, Phosphoinositide-dependent_kinase-1,LMOD3, TTC35, RRP12, ATXN2, ACSM3, SOAT1, FGF8, HNRPH3, CTAGES, POLG2,DYRK3, POLK, Cyclin-dependent_kinase_inhibitor 1C, CD137, Calmodulin_1,ZNF571, CNOT2, CRYZL1, SMC3, SMC4, SLC36A1, Decorin, HKR1, ERC1, S100A6,RIMS1, TMEM67, Mitochondrial_ribosomal_protein_L42, MECP2, RNF111,SULT1A1, MYLK3, TINAG, PRKAR1A, RGPD5, UBE2V1, SAR1B, SLC27A6, ZNF638,RAB33A, TRIOBP, MUCL1, CADPS2, MCF2L, TBCA, SLC17A3, LEO1, IFNA21,RUNX1T1, PRKD2, ATP11B, MORC2, RBM6, KLRD1, MED31, PPHLN1, HMGB2,DNA_repair_and_recombination_protein_RAD54-like, RBM9′, ARL11, HuD,SPEF2, CBLL1, SLC38A1, ‘Caspase_1’, S100G, CA1, CELA1, PTS, ITM2B,Natriuretic_peptide_precursor_C, TRPP3, IMPDH2, DPYS, CDCA3, EFCAB6,SLIT2, SIPA1L1, FIP1L1, ATP6V1B2, HSD17B4, HSD17B7, NDUFC1, CROP, CD48,APPBP1, CD44, CD46, Histone_deacetylase_2_type_XI, Interleukin_4,Tricho-rhino-phalangeal_syndrome_Type_1, SEC61G, TRIP12, PLEKHO1,SEC61B, ST6GALNAC1, CPVL, E2F7, UTP20, E2F5, PARD3, EXOC7, HEXB,Caspase_recruitment_domain-containing_protein 8, MBD4, PPP4C, Helicase,Phosducin, SPG11, CGGBP1, PSKH1, Cathepsin_S, orexin, IMMP2L, C2orf28,Laminin, EIF3S6, LRRC41_type_XII, Cathepsin_C, HPS6, ARAF,Zinc_finger_and_BTB_domain-containing_protein 16,Sex_hormone-binding_globulin, FBLN2, Suppressor_of_cytokine_signaling 1,TMEM126A, DOM3Z, TSFM POLQ-like, DYNLT3, CDH9, EAF2, MIPEP, NDUFA12,HDAC8, MKKS, FGG, IL36G, CDCA7, CRISPLD2, Olfactomedin-like_2b, MRPL32,MRPL33, AHI1, SMARCAL1, UTP14A, SSH2, Dystonin, Contactin_6, PPFIBP1,THOC1, CNOT1, RHCE, SLC41A3, SLC2A9, SNAP23, RFX3, GNG4, MRPL40, LSR,Angiogenin, TRIP4, VRK1, COUP-TFII, FOXP2, SNX2, Nucleoporin_85, RPL37A,RPL27A, SEC62, Calcium-activated_potassium_channel_subunit_alpha-1,SMARCE1, RPL17, CEP104, CEP290, VPS29, ANXA4, Zinc_finger_protein 737,DDX59, SAP30, NEK3, Exosome_component_9,Receptor_for_activated_C_kinase_1, Peptidylprolyl_isomerase_A, TINP1,CEACAM1, DISC1, LRRTM1, POP1_Lamin_B1,SREBP_cleavage-activating_protein, COX6C, TLR_1, ARID2, LACTB, MMS22L,UBE2E3, DAP3, ZNF23, SKP2, GPR113, IRF9 Ghrelin_O-acyltransferase,NEIL3, EEF1E1, COX17, ESD_, Dentin_sialophosphoprotein, HDAC9, RFC4,CYLD, RPLPO, EIF2B3, UGT2A1, FABP7, TRIP11, PLA2G4A, AKR1C3, INTS12,MYH1, ZBTB17, MYH4, NLRP2, MECOM, MYH8, Thermogenin_receptor_2, IFI16,THYN1, RAB17, ETFA, Cystic_fibrosis_transmembrane_conductance_regulator,F13B, RAB6A, ST8SIA1, SATB2, SATB1, HMG20B, UHRF1, CNOT3,Prostaglandin_EP2_receptor, FAM65B,Peroxisome_proliferator-activated_receptor_gamma, KvLQT2, GRIK5, SHOC2,Cortactin, FANCI, KIAA1199, Kynureninase, Decoy_receptor_1, NEU3, PHF10,Methyl-CpG-binding_domain_protein_2, RABGAP1, CEP55, SF3B1, MSHS, MSH6,CREB-binding_protein, LIMS1, SLC5A4, CCNB1IP1, RNF34, SORBS2, UIMC1,SOX5, YWHAZ, ICOSLG, NOP58, Zinc_finger_protein_679, PHKB, MED13, ABCB7,COQ9, C14orf104, Zinc_finger_protein_530, KLRC2, LSM8, NBR1, PRKCD,Long-chain-aldehyde_dehydrogenase, MTSS1, Somatostatin,Ubiquitin_carboxyl-terminal_hydrolase_L5, WDR72, FERMT3,Nuclear_receptor_related-1_protein, Citrate_synthase, VPS11, KIZ,ZFYVE27, BCKDHB, Hypocretin, CACNG2, PTCH1, Carbonic_anhydrase_4,Nucleoporin_107, LDL_receptor, LEKTI, FBXO11, NDUFB3, FCHO2, CEP78,RAPGEF6, PPIL3, NIN, RAPGEF2, Growth_hormone_1, Growth_hormone_2, MNAT1,NAV1, MAP3K8, SUGT1, LAIR1, Hyaluronan-mediated_motility_receptor,MAP3K2, MPP2, TFB2M, CRB3, MPP5, CACNA1G, DLGAP2, INHBA, MAGI2, CIP29,SETDB1, Cytochrome_b5, TRPV2, Interleukin_1_receptor, HOXD8, TIMM10,ATXN2L, CLCN2, CREB1, TNIP1, CBLB, Factor_V, USP33, SON, RBBP8,SLC22A18, PTPN12, ADCY8, MYLK, KIF23, REXO2, BST1, TOP3B, COPB1, AXIN2,COPB2, TNRC6B, Guanidinoacetate_N-methyltransferase,Acyl-CoA_thioesterase_9, C4orf21, TSHB, FRS3, EPB41, Cyclin_T2, LAIR2,Nucleoporin_43, APLP2, TNFRSF19, Death-associated_protein_6,Epithelial_cell_adhesion_molecule, CLEC7A, Gephyrin, CLDND1, VPS37A,PCDHAC2, Bone_morphogenetic_protein_4, NVL, RBM33, RNF139,Sperm_associated_antigen_5, PLCB1,Glial_cell_line-derived_neurotrophic_factor, PARP4, PARP1, MAN2A1,Bone_morphogenetic_protein_1, PAX4, BCCIP, MMPI, Decoy_receptor_3,RAMP2, NCAPD3, LRRC37A, RWDD3, UBE2A, UBE2C, SLC3A1, MRPS22, CDC14A,ITSN1, POLE2, MYC-induced_nuclear_antigen, TMLHE,Glutamate_carboxypeptidase_II, GPR177, PPP2R5C, KIAA1333, RPP38, MYO1F,Farnesoid_X_receptor, Caldesmon, FBXO4, FBXO5, OPN1MW, PIGN, ARNTL2,BCAS3, C6orf58, PHTF2, SEC23A, NUFIP2, OAZ1, Osteoprotegerin, ANAPC4,ATP6V0A2, SPAM1, PSMA6, TAS2R30, RABEP1, DPM3, SLC6A15, RPS26, RPS27,RPS24, RPS20, RPS21, ARHGAP24, Catechol-O-methyl transferase, ERCC5,Transcription_initiation_protein_SPT3_homolog, OR1E1, ZNRF1, GMEB1,CCT2_GNAQ, Mucin_6, Mucin_4, LRP5, PDE9A, C2orf3, EZH2,Epidermal_growth_factor_receptor, TMTC2, PDE4A, EPH_receptor_A4, PPIB,DENND4A, ANTXR1, ANTXR2, Nucleoporin_88, SLCO1B3, COG8, RBMS1, MAP7,HIST2H2BE, AEBP2, DCLRE1A, RPL24, HNRPA2B1, RPL21, RPL23, MAPKAP1,NIPBL, ATG7, SERPINI2, GYLTL1B, ATP5G2, DIP2A, AMY2A, CEP63, TDRD7,PIEZO1, CLDN20, GRXCR1, PMEL, NIF3L1, MCC_, PCNX, TMBIM4, DUSP12,ZMYND8, GOSR1, Interferon_gamma_receptor_1, LDB3, PON3, C1D, ABCC8,COQ7, COQ6, AMELY, HAVCR1, PICALM, Sjogren_syndrome_antigen_B, PLK4,HBB, AKT1, PCDHGB7, C6orf10, UBR1, Retinoblastoma-like_protein 1, GRK6,WWC2, GRK4, INPP4B, SLC34A1, GOLGA2, MYCBP2, PTP4A2, NUCB2, MAGOH,RPP40, Alpha-2A_adrenergic_receptor, SPAG11B, Nucleoporin_205, COG1,Motile_sperm_domain_containing_3, KCNMB3,Motile_sperm_domain_containing_1, KLHL7, KCNN2, TSPAN8, GPR21,Translocator_protein, HNRNPLL, ABHDS, CAB39L, Amphiregulin, GPR1,Interleukin_18, EIF4G3, Interleukin_15, CCDC80, CD2AP, NFS1, GRB2,ULBP2, Vascular_endothelial_growth_factor_C, RPS3, TLR8,BCL2-related_protein_A1, RHOT1, Collagen, Centromere_protein_E, STMN2,HESX1, RPL7, Kalirin, PCMT1, HLA-F, SUMO2, NOX3, EP400, DNM3, EED,NGLY1, NPRL2, PLAC1, Baculoviral_IAP_repeat-containing_protein_3,C7orf31, TUBA1C, HAUS3, IFNA10, MYST4, DCHS1, SIRT4, EFEMP1, ARPC2,MED30, IFT74, PAK1IP1, DYNC1LI2, POLR2B, POLR2H, KIF3A, PRDM16, PLSCR5,PEX5, Parathyroid_hormone_1_receptor, CDC23, RBPMS, MAST1, NRD1, BAT5,BAT2, Dock11, GCSH, POF1B, USP15, POT1, MUTYH, CYP2E1, FAM122C,A1_polypeptide, Flavin_containing_monooxygenase_3, HPGD, LGALS13,MTHFD2L, Survival_motor_neuron_domain_containing_1, PSMA3, MRPS35,MHC_class_I_polypeptide-related_sequence_A, SGCE, REPS1, PPP1R12A,PPP1R12B, PABPC1, MAPK8, PDCD5, Phosphoglucomutase_3, Ubiquitin_C,GABPB2, Mitochondrial_translational_release_factor_1, PFDN4, NUB1,SLC13A3, ZFP36L1, Galectin-3, CC2D2A, GCA,Tissue_factor_pathway_inhibitor, UCKL1, ITFG3, SOS1, WWTR1, GPR84,HSPA14, GJC3, TCF7L1, Matrix_metallopeptidase_12, ISG20, LILRA3,Serum_albumin, Phosducin-like, RPS13, UTP6, HP1BP3, IL12A,HtrA_serine_peptidase_2, LATS1, BMF_, Thymosin_beta-4, B-cell_linker,BCL2L11, Coagulation_factor_XIII, BCL2L12, PRPF19, SFRS5,Interleukin_23_subunit_alpha, NRAP, 60S ribosomal_protein_L14, C9orf64,Testin, VPS13A, DGKD, PTPRB, ATP5C1, KCNJ16, KARS, GTF2H2, AMBN, USP13,ADAMTSL1, TRO_, RTF1, ATP6V1C2, SSBP1,SNRPN_upstream_reading_frame_protein, RPS29, SNRPG, ABCC10, PTPRU,APPL1, TINF2, TMEM22, UNC45A, RPL30, PCDH7, Galactosamine-6_sulfatase,UPF3A, ACTL6A, ACTL6B, IL3RA, SDHB, Cathepsin_L2, TAS2R7, Cathepsin_L1,Pituitary_adenylate_cyclase-activating_peptide, RPN2, DYNLL1, KLK13,NDUFB3, PRPF8, SPINT2, AHSA1, Glutamate_carboxypeptidase_II, DRAP1,RNASE1, Olfactomedin-like_2b, VRK1, IKK2, ERGIC2, TAS2R16, CAMK2G,CAMK2B, Estrogen_receptor_beta, NADH_dehydrogenase, RPL19, NUCB2,KCTD13, ubiquinone, H2AFY, CEP290, PABPC1, HLA-F, DHX38, KIAA0922,MPHOSPH8, DDX59, MIB2_, ZBP1, C16orf84, UACA, C6orf142, MRPL39,Cyclin-dependent_kinase_7, Far_upstream_element-binding_protein_1,SGOL1, GTF2IRD1, ATG10, Dermcidin, EPS8L2, Decorin,Nicotinamide_phosphoribosyltransferase, CDC20, MYB, WNTSA, RBPJ,DEFB103A, RPS15A, ATPSH, RPS3, FABP1, SLC4A8, Serum_amyloid_P_component,ALAS1, MAPK1, PDCD5, SULT1A1, CHRNA3, ATXN10, MNAT1, ALG13, Ataxin_3,LRRC39, ADH7, Delta-sarcoglycan, TACC1, IFNA4,Thymic_stromal_lymphopoietin, LGTN, KIAA1333, MSH6, MYOT, RIPKS,BCL2L11, RPL27, Rnd1, Platelet_factor 4, HSD17B7, LSM8, CEP63, INTS8,CTNS, ASAHL, CELA3A, SMARCAL1, HEXB, SLC16A5, MAP3K12, FRMD6.

The RDE can be a Class I AU rich element that arises from the 3′ UTR ofa gene encoding, for example, c-myc, c-fos, betal-AR, PTH,interferon-gamma, MyoD, p21, Cyclin A, Cyclin B1, Cyclin D1, PAI-2, orNOS HANOS. The RDE can also be a Class II AU rich element and arisesfrom the 3′ UTR of a gene encoding, for example, GM-CSF, TNF-alpha,interferon-alpha, COX-2, IL-2, IL-3, bcl-2, interferon-beta, or VEG-F.The RDE can be a Class III AU rich element that arises from the 3′ UTRof a gene encoding, for example, c-jun, GLUT1, p53, hsp 70, Myogenin,NF-M, or GAP-43. Other RDEs may be obtained from the 3′-UTRs of a T-cellreceptor subunit (α, β, γ, or δ chains), cytotoxicT-lymphocyte-associated antigen 4 (CTLA4), programmed cell death protein(PD-1), Killer-cell Immunoglobulin-like Receptors (KIR), and LymphocyteActivation Gene-3 (LAG3), and other checkpoint inhibitors. Still otherRDEs may be obtained from the 3′-UTRs of senescence-associated secretoryphenotype genes disclosed in Coppe et al., Ann. Rev. Pathol. 5:99-418(2010), which is incorporated by reference in its entirety for allpurposes (e.g., see Table 1).

The RDE can be bound by certain polypeptides including, for example, AREpoly(U) binding/degradation factor (AUF-1), tristetraprolin (TTP), humanantigen-related protein (HuR), butyrate response factor 1 (BRF-1),butyrate response factor 2 (BRF-2), T-cell restricted intracellularantigen-1 (TIA-1), TIA-1 related protein (TIAR), CUG triplet repeat, RNAbinding protein 1 (CUGBP-1), CUG triplet repeat, RNA binding protein 2(CUGBP-2), human neuron specific RNA binding protein (Hel-N1, Hel-N2),RNA binding proteins HuA, HuB and HuC, KH-type splicing regulatoryprotein (KSRP), 3-methylglutaconyl-CoA hydratase (AUH), glyceraldehyde3-phosphate dehydrogenase (GAPDH), heat shock protein 70 (Hsp70), heatshock protein 10 (Hsp10), heterogeneous nuclear ribonucleoprotein A1(hnRNP A1), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2),heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3), heterogeneousnuclear ribonucleoprotein C (hnRNP C), heterogeneous nuclearribonucleoprotein L (hnRNP L), Bcl-2 AU-rich element RNA binding protein(TINO), Poly(A) Binding Protein Interacting Protein 2 (PAIP2), IRP1,pyruvate kinase, lactate dehydrogenase, enolase, and aldolase. The RDEbinding protein also can be an enzyme involved in glycolysis orcarbohydrate metabolism, such as, for example, Glyceraldehyde PhosphateDehydrogenase (GAPDH), enolase (ENO1 or ENO3), Phosphoglycerate Kinase(PGK1), Triosephosphate Isomerase (TPI1), Aldolase A (ALDOA),Phosphoglycerate Mutase (PGAM1), Hexokinase (HK-2), or LactateDehydrogenase (LDH). The RDE binding protein can be an enzyme involvedin the Pentose Phosphate Shunt, including for example, Transketolase(TKT) or Triosephosphate Isomerase (TPI1). Additional exemplary RNAbinding proteins are those described in Castello et al., Molc. Cell63:696-710 (2016); Kovarik et al., Cytokine 89:21-26 (2017); Ray et al.,Nature 499:172-177 (2013); Castello et al., Cell 149:1393-1406 (2012);Vlasova et al., Molc. Cell. 29:263-270 (2008); Barreau et al., Nucl.Acids Res. vol 33, doi:10.1093/nar/gki1012 (2006); Meisner et al.,ChemBioChem 5:1432-1447 (2004); Guhaniyogi et al., Gene 265:11-23(2001), all of which are incorporated by reference in their entirety forall purposes.

The RDE binding protein can be TTP which can bind to RDEs including forexample, one or more of UUAUUUAUU (SEQ ID NO: 19) and AUUUA (SEQ ID NO:8), or KSRP which binds AU-rich RDEs, or Auf1 which binds RDEs includingfor example, one or more of UUGA (SEQ ID NO: 20), AGUUU (SEQ ID NO: 18),or GUUUG (SEQ ID NO: 12), or CELF-1 which binds RDEs including forexample, one or more of UUGUU (SEQ ID NO: 10), or HuR which binds RDEsincluding for example, one or more of UUUAUUU (SEQ ID NO: 15), UUUUUUU(SEQ ID NO: 16), or UUUGUUU (SEQ ID NO: 13), or ESRP1 or ESRP2 whichbinds RDEs including for example, one or more of UGGGGAU (SEQ ID NO:21), or ELAV which binds RDEs including for example, one or more ofUUUGUUU (SEQ ID NO: 13). The RDE binding protein can be an enzymeinvolved in glycolysis, including for example, GAPDH which binds AU richelements including for example, one or more of AUUUA (SEQ ID NO: 8)elements, or ENO3/ENO1 which binds RDEs including for example, one ormore of CUGCUGCUG (SEQ ID NO: 22), or ALDOA which binds RDEs includingfor example, one or more of AUUGA (SEQ ID NO: 23).

Some RNA binding proteins increase the rate of RNA degradation afterbinding to the RDE. Some RNA binding proteins decrease the rate ofdegradation of the RNA after binding to the RDE. More than one RNAbinding protein binds can bind to an RDE. In some RDE regulatory units,more than one RNA binding protein binds to more than one RDE. Binding ofone or more of the RNA binding proteins to the one or more RDEs canincrease the degradation rate of the RNA. Binding of one or more of theRNA binding proteins can decrease the degradation rate of the RNA. RNAbinding proteins that increase degradation may compete for binding to anRDE with RNA binding proteins that decrease degradation, so that thestability of the RNA is dependent of the relative binding of the two RNAbinding proteins. Other proteins can bind to the RDE binding proteinsand modulate the effect of the RNA binding protein on the RNA with theRDE. Binding of a protein to the RNA binding protein can increases RNAstability or decrease RNA stability. An RNA can have multiple RDEs thatare bound by the proteins HuR and TTP. The HuR protein can stabilize theRNA and the TTP protein can destabilize the RNA. An RNA can have atleast one RDE that interacts with the proteins KSRP, TTP and/or HuR.KSRP can destabilize the RNA and compete for binding with the HuRprotein that can stabilize the RNA. The KSRP protein can bind to the RDEand destabilizes the RNA and the TTP protein can bind to KSRP andprevent degradation of the RNA. Different proteins may be bound to thesame transcript and may have competing effects on degradation andstabilization rates. Different proteins may be bound to the sametranscript and may have cooperative effects on degradation andstabilization rates. Different proteins may be bound to the sametranscript at different times, conferring different effects ondegradation and stabilization.

The RDE can be a Class II AU rich element, and the RNA binding proteincan be GAPDH. The Class II AU rich element bound by GAPDH can beAUUUAUUUAUUUA (SEQ ID NO: 9). The Class II AU rich element and GADPH canbe used to control the expression of a transgene, a CAR, Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side-CAR. The Class II AU rich element andGADPH also can be used to effect the expression of a transgene, CAR,Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR in a T-lymphocyte. TheClass II AU rich element and GADPH can be used to effect the expressionof a transgene, CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR ina CD8+ T-lymphocyte. The Class II AU rich element and GADPH can be usedto effect the expression of a transgene, CAR, Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side-CAR in a CD4+ T-lymphocyte. The Class II AUrich element and GADPH can be used to effect the expression of atransgene, CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR in anatural killer cell.

The RDE may have microRNA binding sites. The RDE can be engineered toremove one or more of these microRNA binding sites. The removal of themicroRNA binding sites can increase the on expression from a constructwith an RDE by at least 5, 10, 15, 20, 50 or 100 fold. The RDE with themicroRNA sites can be an RDE that is bound by GAPDH. The removal ofmicroRNA sites from the RDE bound by GAPDH can increase the onexpression of a construct with the GAPDH sensitive RDE by at least 5-10fold. This GAPDH control through the RDE can be used to deliver apayload at a target site. The GAPDH control can be tied to activation ofthe eukaryotic cell by a CAR that recognizes an antigen foundpreferentially at the target site.

The RDE can be the 3′-UTR of IL-2 or IFN-γ, and removal of micro-RNAsites can increase the rate of expression and/or the dynamic range ofexpression from a transgene RNA with the RDE. The RDE can be the 3′-UTRof IL-2 and the removed micro-RNA sites can be the MIR-186 sites whichdeletion increases the kinetics of expression and increases the dynamicrange of expression by about 50-fold. The RDE also can be the 3′-UTR ofIFN-γ and the micro-RNA sites removed can be the MIR-125 sites.

Chimeric Antigen Receptors

Chimeric antigen receptors (CARs) can be fused proteins comprising anextracellular antigen-binding/recognition element, a transmembraneelement that anchors the receptor to the cell membrane and at least oneintracellular element. These CAR elements are known in the art, forexample as described in patent application US20140242701, which isincorporated by reference in its entirety for all purposes herein. TheCAR can be a recombinant polypeptide expressed from a constructcomprising at least an extracellular antigen binding element, atransmembrane element and an intracellular signaling element comprisinga functional signaling element derived from a stimulatory molecule. Thestimulatory molecule can be the zeta chain associated with the T cellreceptor complex. The cytoplasmic signaling element may further compriseone or more functional signaling elements derived from at least onecostimulatory molecule. The costimulatory molecule can be chosen from4-1BB (i.e., CD137), CD27 and/or CD28. The CAR may be a chimeric fusionprotein comprising an extracellular antigen recognition element, atransmembrane element and an intracellular signaling element comprisinga functional signaling element derived from a stimulatory molecule. TheCAR may comprise a chimeric fusion protein comprising an extracellularantigen recognition element, a transmembrane element and anintracellular signaling element comprising a functional signalingelement derived from a co-stimulatory molecule and a functionalsignaling element derived from a stimulatory molecule. The CAR may be achimeric fusion protein comprising an extracellular antigen recognitionelement, a transmembrane element and an intracellular signaling elementcomprising two functional signaling elements derived from one or moreco-stimulatory molecule(s) and a functional signaling element derivedfrom a stimulatory molecule. The CAR may comprise a chimeric fusionprotein comprising an extracellular antigen recognition element, atransmembrane element and an intracellular signaling element comprisingat least two functional signaling elements derived from one or moreco-stimulatory molecule(s) and a functional signaling element derivedfrom a stimulatory molecule. The CAR may comprise an optional leadersequence at the amino-terminus (N-term) of the CAR fusion protein. TheCAR may further comprise a leader sequence at the N-terminus of theextracellular antigen recognition element, wherein the leader sequenceis optionally cleaved from the antigen recognition element (e.g., ascFv) during cellular processing and localization of the CAR to thecellular membrane.

Chimeric Antigen Receptor—Extracellular Element

Exemplary extracellular elements useful in making CARs are described,for example, in U.S. patent application Ser. No. 15/070,352 filed onMar. 15, 2016, and U.S. patent application Ser. No. 15/369,132 filedDec. 5, 2016, both of which are incorporated by reference in theirentirety for all purposes.

The extracellular element(s) can be obtained from the repertoire ofantibodies obtained from the immune cells of a subject that has becomeimmune to a disease, such as for example, as described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, both of which areincorporated by reference in their entirety for all purposes.

The extracellular element may be obtained from any of the wide varietyof extracellular elements or secreted proteins associated with ligandbinding and/or signal transduction as described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, U.S. Pat. Nos.5,359,046, 5,686,281 and 6,103,521, all of which are incorporated byreference in their entirety for all purposes.

As described in U.S. patent application Ser. No. 15/070,352 filed onMar. 15, 2016, and U.S. patent application Ser. No. 15/369,132 filedDec. 5, 2016, both of which are incorporated by reference in theirentirety for all purposes, there is provided a Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CAR capable of binding to an antigens derived from viruses,infectious diseases, bacteria, tumor associated antigens, inflammatorydisease associated antigens, antigens associated with neuronaldisorders, antigens associated with diabetes, antigens associated withsenescent cells, antigens associated with cardiovascular diseases,antigens associated with autoimmune diseases, and/or antigens associatedwith allergies. Examples of antigens useful for these applications arefound, for example, in U.S. patent application Ser. No. 15/070,352 filedon Mar. 15, 2016, and U.S. patent application Ser. No. 15/369,132 filedDec. 5, 2016, both of which are incorporated by reference in theirentirety for all purposes.

Intracellular Element

The intracellular element can be a molecule that can transmit a signalinto a cell when the extracellular element of the Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CAR binds to (interacts with) an antigen. The intracellularsignaling element can be generally responsible for activation of atleast one of the normal effector functions of the immune cell in whichthe Smart CAR(s), DE-CAR(s), RDE-CAR(s), Smart-RDE-CAR(s),DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s) and/or Side-CAR(s)has been introduced. The term “effector function” refers to aspecialized function of a cell. Effector function of a T cell, forexample, may be cytolytic activity or helper activity including thesecretion of cytokines. Thus the term “intracellular signaling element”refers to the portion of a protein which transduces the effectorfunction signal and directs the cell to perform a specialized function.While the entire intracellular signaling domain can be employed, in manycases the intracellular element or intracellular signaling element neednot consist of the entire domain. To the extent that a truncated portionof the intracellular signaling domain is used, such truncated portionmay be used as long as it transduces the effector function signal. Theterm intracellular signaling element is thus also meant to include anytruncated portion of the intracellular signaling domain sufficient totransduce the effector function signal. Examples of intracellularsignaling elements for use in the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR of the invention include the cytoplasmic sequences of the Tcell receptor (TCR) and co-receptors that act in concert to initiatesignal transduction following antigen receptor engagement, as well asany derivative or variant of these sequences and any recombinantsequence that has the same functional capability.

Intracellular elements and combinations of polypeptides useful with oras intracellular elements are described, for example, in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, both of which areincorporated by reference in their entirety for all purposes.

Transmembrane Element and Spacer Element

The Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR may comprise a transmembrane element.The transmembrane element can be attached to the extracellular elementof the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR. The transmembrane elementcan include one or more additional amino acids adjacent to thetransmembrane region, e.g., one or more amino acid associated with theextracellular region of the protein from which the transmembrane wasderived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of theextracellular region) and/or one or more additional amino acidsassociated with the intracellular region of the protein from which thetransmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 upto 15 amino acids of the intracellular region). The transmembraneelement can be associated with one of the other elements used in theSmart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR. The transmembrane element can beselected or modified by amino acid substitution to avoid binding of suchelements to the transmembrane elements of the same or different surfacemembrane proteins, e.g., to minimize interactions with other members ofthe receptor complex. The transmembrane element can be capable ofhomodimerization with another Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR on the cellsurface. The amino acid sequence of the transmembrane element may bemodified or substituted so as to minimize interactions with the bindingelements of the native binding partner present in the same cell.

The transmembrane element may be contributed by the protein contributingthe multispecific extracellular inducer clustering element, the proteincontributing the effector function signaling element, the proteincontributing the proliferation signaling portion, or by a totallydifferent protein. For the most part it will be convenient to have thetransmembrane element naturally associated with one of the elements. Insome cases it will be desirable to employ the transmembrane element ofthe ζ, η or FcεR1γ chains which contain a cysteine residue capable ofdisulfide bonding, so that the resulting chimeric protein will be ableto form disulfide linked dimers with itself, or with unmodified versionsof the ζ, η or FcεR1γ chains or related proteins. The transmembraneelement can be selected or modified by amino acid substitution to avoidbinding of such elements to the transmembrane elements of the same ordifferent surface membrane proteins to minimize interactions with othermembers of the receptor complex. The transmembrane element of ζ, η,FcεR1-γ and -β, MB1 (Igα), B29 or CD3-γ, ζ, or ε, may be used in orderto retain physical association with other members of the receptorcomplex.

Transmembrane elements useful in the present invention are described,fore example, in U.S. patent application Ser. No. 15/070,352 filed onMar. 15, 2016, and U.S. patent application Ser. No. 15/369,132 filedDec. 5, 2016, both of which are incorporated by reference in theirentirety for all purposes.

Chimeric Antigen Receptors Coupled with Destabilizing Elements (DE-CAR)

Destabilizing elements, as described above, can be combined in cis witha CAR, as described above, so that the amount of the CAR polypeptide inthe eukaryotic cell is under the control of the DE. DE-CARs, selectionof DEs, and use of one or multiple DEs is described, for example, inU.S. patent application Ser. No. 15/070,352 filed on Mar. 15, 2016, andU.S. patent application Ser. No. 15/369,132 filed Dec. 5, 2016, both ofwhich are incorporated by reference in their entirety for all purposes.

Chimeric Antigen Receptors: Side-CARs

The CARs, Smart CARs, DE-CARs, RDE-CARs, Smart-RDE-CARs, DE-RDE-CARs,Smart-DE-CARs, and/or Smart-DE-RDE-CARs can be comprised of at least twoparts which associate to form a functional CAR or DE-CAR. Theextracellular antigen binding element can be expressed as a separatepart from the transmembrane element, optional spacer, and theintracellular element of a CAR. The separate extracellular bindingelement can be associated with the host cell membrane (through a meansother than a transmembrane polypeptide). The intracellular element canbe expressed as a separate part from the extracellular element,transmembrane element, and optionally the spacer. The extracellularelement and intracellular element can be expressed separately and eachcan have a transmembrane element, and optionally a spacer. Each part ofthe CAR or DE-CAR can have an association element (“Side-CAR”) forbringing the two parts together to form a functional CAR or DE-CAR.

Side CARs, selection of Side CARs, and their use with or without atether are described, for example, in U.S. patent application Ser. No.15/070,352 filed on Mar. 15, 2016, and U.S. patent application Ser. No.15/369,132 filed Dec. 5, 2016, both of which are incorporated byreference in their entirety for all purposes.

Lymphocyte Expansion Molecule and Other Regulatory Factors

The use of DEs, RDEs, and/or RNA control devices to control expressionof lymphocyte expansion molecule (“LEM”), IL1, IL2, IL4, IL5, IL6, IL7,IL10, IL12, IL15, GM-CSF, G-CSF, TNFα, and/or IFNγ is described, forexample, in U.S. patent application Ser. No. 15/070,352 filed on Mar.15, 2016, and U.S. patent application Ser. No. 15/369,132 filed Dec. 5,2016, both of which are incorporated by reference in their entirety forall purposes.

Dominant Negative Regulators as CAR Off-Switches

A control device may regulate the expression of a polypeptide thatinhibits or reduces the ability of a CAR to activate a T-lymphocyte ornatural killer cell. This polypeptide can inhibit or reduce CARactivation, such as, for example, a mutant of ZAP-70 that has adominant-negative effect when expressed in T-lymphocytes. Such ZAP-70mutant can be a Δ277-619 (leaving residues 1-276), Y319F, or K369Amutant of ZAP-70.

In some aspects, dominant-negative mutants are used to turn offsignaling from an activated CAR at desired times. The dominant-negativemutants are polypeptides that disrupt the intracellular signaling froman activated CAR. Dominant-negative mutants can be used frompolypeptides from or that interact with the signaling cascade activatedby the CAR. Such mutants may interact with some signaling components butare defective for propagating the signal from the CAR and so preventfurther activation of the host cell by the CAR. The dominant-negativemutant can be derived from the ZAP 70 protein. The dominant-negativemutant may be a Δ277-619 (leaving residues 1-276) of ZAP 70, Y319F ofZAP 70, or K369A of ZAP 70.

The expression of the dominant-negative mutant can be under theinducible control of an RNA control device, an RDE, a destabilizingelement (DE), and/or an inducible promoter. This inducible controlallows expression of the dominant-negative mutant at a desired timeallowing this construct to act as an off switch for a CAR eukaryoticcell. At a desired time, the dominant-negative mutant is expressed inresponse to the inducing stimulus, and the dominant-negative mutantturns off signaling from the CAR, DE-CAR and/or Side-CAR.

A Tet RNA control device or a (6R)-folinic acid RNA control device asdescribed above can be used to control the expression of adominant-negative mutant of ZAP 70 (e.g., a Δ277-619 (leaving residues1-276) mutant of Zap 70, Y319F ZAP 70, or K369A of ZAP 70). With theseconstructs, the dominant-negative mutant of Zap 70 can be expressed in ahost cell upon addition of tetracycline or (6R)-folinic acid to themedia. This tetracycline or (6R)-folinic acid RNA control deviceprovides an off-switch for CAR activity that is inducible bytetracycline or (6R)-folinic acid.

The dominant-negative mutant of ZAP-70 can be placed under the controlof a suitable RDE, DE, RNA control device, or Side-CAR. The RNA controldevice can be a Tet or (6R)-folinic acid reactive RNA control device sothat when control device ligand (e.g., Tet or (6R)-folinic acid) isintroduced the dominant-negative ZAP-70 mutant is expressed, and theexpression of the ZAP-70 mutant inhibits activation of the T-lymphocyteby the CAR. The ZAP-70 mutant can act as an off-switch for theT-lymphocyte that is under the control of the Tet control device.

Receptors

CARs may be used as the receptor with the cell and the RDE-transgene.CARs are described above. In addition to CARs, other receptors may beused to activate or otherwise change conditions in a cell so that atransgene under the control of an RDE is expressed. Receptors thatrecognize and respond to a chemical signal can be coupled to expressionof the transgene through the RDE. For example, ion channel-linked(ionotropic) receptors, G protein-linked (metabotropic) receptors, andenzyme-linked receptors can be coupled to the expression of thetransgene.

One class of receptor that can be coupled to transgene expression areimmune receptors such as, for example, T-cell receptors, B-cellreceptors (aka antigen receptor or immunoglobulin receptor), and innateimmunity receptors.

T-cell receptors are heterodimers of two different polypeptide chains.In humans, most T cells have a T-cell receptor made of an alpha (α)chain and a beta (β) chain have a T-cell receptor made of gamma anddelta (γ/δ) chains (encoded by TRG and TRD, respectively). Techniquesand primers for amplifying nucleic acids encoding the T-cell receptorchains from lymphocytes are well known in the art and are described in,for example, SMARTer Human TCR a/b Profiling Kits sold commercially byClontech, Boria et al., BMC Immunol. 9:50-58 (2008); Moonka et al., J.Immunol. Methods 169:41-51 (1994); Kim et al., PLoS ONE 7:e37338 (2012);Seitz et al., Proc. Natl Acad. Sci. 103:12057-62 (2006), all of whichare incorporated by reference in their entirety for all purposes. TheTCR repertoires can be used as separate chains to form an antigenbinding domain. The TCR repertoires can be converted to single chainantigen binding domains. Single chain TCRs can be made from nucleicacids encoding human alpha and beta chains using techniques well-knownin the art including, for example, those described in U.S. PatentApplication Publication No. US2012/0252742, Schodin et al., Mol.Immunol. 33:819-829 (1996); Aggen et al., “Engineering HumanSingle-Chain T Cell Receptors,” Ph.D. Thesis with the University ofIllinois at Urbana-Champaign (2010) a copy of which is found atideals.illinois.edu/bitstream/handle/2142/18585/Aggen_David.pdf?sequence=1,all of which are incorporated by reference in their entirety for allpurposes.

B-cell receptors include an immunoglobulin that is membrane bound, asignal transduction moiety, CD79, and an ITAM. Techniques and primersfor amplifying nucleic acids encoding human antibody light and heavychains are well-known in the art, and described in, for example,ProGen's Human IgG and IgM Library Primer Set, Catalog No. F2000;Andris-Widhopf et al., “Generation of Human Fab Antibody Libraries: PCRAmplification and Assembly of Light and Heavy Chain Coding Sequences,”Cold Spring Harb. Protoc. 2011; Lim et al., Nat. Biotechnol. 31:108-117(2010); Sun et al., World J. Microbiol. Biotechnol. 28:381-386 (2012);Coronella et al., Nucl. Acids. Res. 28:e85 (2000), all of which areincorporated by reference in their entirety for all purposes. Techniquesand primers for amplifying nucleic acids encoding mouse antibody lightand heavy chains are well-known in the art, and described in, forexample, U.S. Pat. No. 8,143,007; Wang et al., BMC Bioinform.7(Suppl):S9 (2006), both of which are incorporated by reference in theirentirety for all purposes. The antibody repertoires can be used asseparate chains in antigen binding domains, or converted to single chainantigen binding domains. Single chain antibodies can be made fromnucleic acids encoding human light and heavy chains using techniqueswell-known in the art including, for example, those described in Pansriet al., BMC Biotechnol. 9:6 (2009); Peraldi-Roux, Methods Molc. Biol.907:73-83 (2012), both of which are incorporated by reference in theirentirety for all purposes. Single chain antibodies can be made fromnucleic acids encoding mouse light and heavy chains using techniqueswell-known in the art including, for example, those described in Imai etal., Biol. Pharm. Bull. 29:1325-1330 (2006); Cheng et al., PLoS ONE6:e27406 (2011), both of which are incorporated by reference in theirentirety for all purposes.

Innate immunity receptors include, for example, the CD94/NKG2 receptorfamily (e.g., NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H), the 2B4receptor, the NKp30, NKp44, NKp46, and NKp80 receptors, the Toll-likereceptors (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, RP105).

G-protein linked receptors also known as seven-transmembrane domainreceptors are a large family of receptors that couple receptor bindingof ligand to cellular responses through G proteins. These G-proteins aretrimers of α, β, and γ subunits (known as Gα, Gβ, and Gγ, respectively)which are active when bound to GTP and inactive when bound to GDP. Whenthe receptor binds ligand it undergoes a conformational change andallosterically activates the G-protein to exchange GTP for bound GDP.After GTP binding the G-protein dissociates from the receptor to yield aGa-GTP monomer and a Gβγ dimer. G-protein linked receptors have beengrouped together into classes which include, for example, Rhodopsin-likereceptors, secretin receptors, metabotropic glutamate/pheromonereceptors, fungal mating pheromone receptors, cyclic AMP receptors, andfrizzled/smoothened receptors. G-protein receptors are used in a widevariety of physiological processes including detection ofelectromagnetic radiation, gustatory sense (taste), sense of smell,neurotransmission, immune system regulation, growth, cell densitysensing, etc.

Enzyme linked receptors also known as a catalytic receptor, is atransmembrane receptor, where the binding of an extracellular ligandcauses enzymatic activity on the intracellular side. Enzyme linkedreceptors have two domains joined together by a transmembrane portion(or domain) of the polypeptide. The two terminal domains are anextracellular ligand binding domain and an intracellular domain that hasa catalytic function. There are multiple families of enzyme linkedreceptors including, for example, the Erb receptor family, the glialcell-derived neurotrophic factor receptor family, the natriureticpeptide receptor family, the trk neurotrophin receptor family, and thetoll-like receptor family.

Ion channel linked receptors also known as ligand-gated ion channels arereceptors that allow ions such as, for example, Na⁺, K⁺, Ca²⁺ and Cl⁻ topass through the membrane in response to the binding of a ligand to thereceptor. There are multiple families of ligand-gated ion channelsincluding, for example, cationic cys-loop receptors, anionic cys-loopreceptors, ionotropic glutamate receptors (AMPA receptors, NMDAreceptors), GABA receptors, 5-HT receptors, ATP-gated channels, andPIP₂-gated channels.

Eukaryotic Cells

Various eukaryotic cells can be used as the eukaryotic cell of theinvention. The eukaryotic cells can be animal cells. The eukaryoticcells can be mammalian cells, such as mouse, rat, rabbit, hamster,porcine, bovine, feline, or canine. The mammalian cells can be cells ofprimates, including but not limited to, monkeys, chimpanzees, gorillas,and humans. The mammalians cells can be mouse cells, as mice routinelyfunction as a model for other mammals, most particularly for humans(see, e.g., Hanna, J. et al., Science 318:1920-23, 2007; Holtzman, D. M.et al., J Clin Invest. 103(6):R15-R21, 1999; Warren, R. S. et al., JClin Invest. 95: 1789-1797, 1995; each publication is incorporated byreference in its entirety for all purposes). Animal cells include, forexample, fibroblasts, epithelial cells (e.g., renal, mammary, prostate,lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, andhematopoietic cells. The animal cells can be adult cells (e.g.,terminally differentiated, dividing or non-dividing) or embryonic cells(e.g., blastocyst cells, etc.) or stem cells. The eukaryotic cell alsocan be a cell line derived from an animal or other source.

The eukaryotic cells can be stem cells. A variety of stem cells typesare known in the art and can be used as the eukaryotic cell, includingfor example, embryonic stem cells, inducible pluripotent stem cells,hematopoietic stem cells, neural stem cells, epidermal neural crest stemcells, mammary stem cells, intestinal stem cells, mesenchymal stemcells, olfactory adult stem cells, testicular cells, and progenitorcells (e.g., neural, angioblast, osteoblast, chondroblast, pancreatic,epidermal, etc.). The stem cells can be stem cell lines derived fromcells taken from a subject.

The eukaryotic cell can be a cell found in the circulatory system of amammal, including humans. Exemplary circulatory system cells include,among others, red blood cells, platelets, plasma cells, T-cells, naturalkiller cells, B-cells, macrophages, neutrophils, or the like, andprecursor cells of the same. As a group, these cells are defined to becirculating eukaryotic cells of the invention. The eukaryotic cell canbe derived from any of these circulating eukaryotic cells. Transgenesmay be used with any of these circulating cells or eukaryotic cellsderived from the circulating cells. The eukaryotic cell can be a T-cellor T-cell precursor or progenitor cell. The eukaryotic cell can be ahelper T-cell, a cytotoxic T-cell, a memory T-cell, a regulatory T-cell,a natural killer T-cell, a mucosal associated invariant T-cell, a gammadelta T cell, or a precursor or progenitor cell to the aforementioned.The eukaryotic cell can be a natural killer cell, or a precursor orprogenitor cell to the natural killer cell. The eukaryotic cell can be aB-cell, or a B-cell precursor or progenitor cell. The eukaryotic cellcan be a neutrophil or a neutrophil precursor or progenitor cell. Theeukaryotic cell can be a megakaryocyte or a precursor or progenitor cellto the megakaryocyte. The eukaryotic cell can be a macrophage or aprecursor or progenitor cell to a macrophage.

The eukaryotic cells can be plant cells. The plant cells can be cells ofmonocotyledonous or dicotyledonous plants, including, but not limitedto, alfalfa, almonds, asparagus, avocado, banana, barley, bean,blackberry, brassicas, broccoli, cabbage, canola, carrot, cauliflower,celery, cherry, chicory, citrus, coffee, cotton, cucumber, eucalyptus,hemp, lettuce, lentil, maize, mango, melon, oat, papaya, pea, peanut,pineapple, plum, potato (including sweet potatoes), pumpkin, radish,rapeseed, raspberry, rice, rye, sorghum, soybean, spinach, strawberry,sugar beet, sugarcane, sunflower, tobacco, tomato, turnip, wheat,zucchini, and other fruiting vegetables (e.g. tomatoes, pepper, chili,eggplant, cucumber, squash etc.), other bulb vegetables (e.g., garlic,onion, leek etc.), other pome fruit (e.g. apples, pears etc.), otherstone fruit (e.g., peach, nectarine, apricot, pears, plums etc.),Arabidopsis, woody plants such as coniferous and deciduous trees, anornamental plant, a perennial grass, a forage crop, flowers, othervegetables, other fruits, other agricultural crops, herbs, grass, orperennial plant parts (e.g., bulbs; tubers; roots; crowns; stems;stolons; tillers; shoots; cuttings, including un-rooted cuttings, rootedcuttings, and callus cuttings or callus-generated plantlets; apicalmeristems etc.). The term “plants” refers to all physical parts of aplant, including seeds, seedlings, saplings, roots, tubers, stems,stalks, foliage and fruits.

The eukaryotic cells also can be algal, including but not limited toalgae of the genera Chlorella, Chlamydomonas, Scenedesmus, Isochrysis,Dunaliella, Tetraselmis, Nannochloropsis, or Prototheca. The eukaryoticcells can be fungi cells, including, but not limited to, fungi of thegenera Saccharomyces, Klyuveromyces, Candida, Pichia, Debaryomyces,Hansenula, Yarrowia, Zygosaccharomyces, or Schizosaccharomyces.

The eukaryotic cells can be obtained from a subject. The subject may beany living organisms. The cells can be derived from cells obtained froma subject. Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T cells can be obtained from a number ofsources, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, ascites, pleural effusion, spleen tissue, and tumors. Anynumber of T cell lines available in the art also may be used. T-cellscan be obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as Ficollseparation. Cells from the circulating blood of an individual can beobtained by apheresis. The apheresis product typically containslymphocytes, including T cells, monocytes, granulocytes, B cells, othernucleated white blood cells, red blood cells, and platelets. The cellscollected by apheresis may be washed to remove the plasma fraction andto place the cells in an appropriate buffer or media for subsequentprocessing steps. The cells can be washed with phosphate buffered saline(PBS). In an alternative aspect, the wash solution lacks calcium and maylack magnesium or may lack many if not all divalent cations. Initialactivation steps in the absence of calcium can lead to magnifiedactivation.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. Cells can be enrichedby cell sorting and/or selection via negative magnetic immunoadherenceor flow cytometry using a cocktail of monoclonal antibodies directed tocell surface markers present on the cells. For example, to enrich forCD4+ cells, a monoclonal antibody cocktail typically includes antibodiesto CD14, CD20, CD11b, CD16, HLA-DR, and CD8. It may be desirable toenrich for regulatory T cells which typically express CD4+, CD25+,CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain aspects, Tregulatory cells are depleted by anti-C25 conjugated beads or othersimilar method of selection.

T cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005, each of which is incorporated by reference in its entiretyfor all purposes.

NK cells may be expanded in the presence of a myeloid cell line that hasbeen genetically modified to express membrane bound IL-15 and 4-1BBligand (CD137L). A cell line modified in this way which does not haveMHC class I and II molecules is highly susceptible to NK cell lysis andactivates NK cells. For example, K562 myeloid cells can be transducedwith a chimeric protein construct consisting of human IL-15 maturepeptide fused to the signal peptide and transmembrane domain of humanCD8a and GFP. Transduced cells can then be single-cell cloned bylimiting dilution and a clone with the highest GFP expression andsurface IL-15 selected. This clone can then be transduced with humanCD137L, creating a K562-mb15-137L cell line. To preferentially expand NKcells, peripheral blood mononuclear cell cultures containing NK cellsare cultured with a K562-mb15-137L cell line in the presence of 10 IU/mLof IL-2 for a period of time sufficient to activate and enrich for apopulation of NK cells. This period can range from 2 to 20 days,preferably about 5 days. Expanded NK cells may then be transduced withthe anti-CD19-BB-ζ chimeric receptor.

Nucleic Acids

Also described in this disclosure are nucleic acids that encode, atleast in part, the individual peptides, polypeptides, proteins, and RNAcontrol devices described herein. The nucleic acids may be natural,synthetic or a combination thereof. The nucleic acids of the inventionmay be RNA, mRNA, DNA or cDNA.

The nucleic acids of the invention also include expression vectors, suchas plasmids, or viral vectors, or linear vectors, or vectors thatintegrate into chromosomal DNA. Expression vectors can contain a nucleicacid sequence that enables the vector to replicate in one or moreselected host cells. Such sequences are well known for a variety ofcells. The origin of replication from the plasmid pBR322 is suitable formost Gram-negative bacteria. In eukaryotic host cells, e.g., mammaliancells, the expression vector can be integrated into the host cellchromosome and then replicate with the host chromosome. Similarly,vectors can be integrated into the chromosome of prokaryotic cells.

Expression vectors also generally contain a selection gene, also termeda selectable marker. Selectable markers are well-known in the art forprokaryotic and eukaryotic cells, including host cells of the invention.Generally, the selection gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli. An exemplary selection schemecan utilize a drug to arrest growth of a host cell. Those cells that aresuccessfully transformed with a heterologous gene produce a proteinconferring drug resistance and thus survive the selection regimen. Otherselectable markers for use in bacterial or eukaryotic (includingmammalian) systems are well-known in the art.

An example of a promoter that is capable of expressing a Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR transgene in a mammalian T cell is theEF1a promoter. The native EF1a promoter drives expression of the alphasubunit of the elongation factor-1 complex, which is responsible for theenzymatic delivery of aminoacyl tRNAs to the ribosome. The EF1a promoterhas been extensively used in mammalian expression plasmids and has beenshown to be effective in driving CAR expression from transgenes clonedinto a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8):1453-1464 (2009), which is incorporated by reference in its entirety forall purposes. Another example of a promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Other constitutive promoter sequences may also be used, including, butnot limited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus promoter (MMTV), human immunodeficiency virus (HIV) longterminal repeat (LTR) promoter, MoMuLV promoter, phosphoglycerate kinase(PGK) promoter, MND promoter (a synthetic promoter that contains the U3region of a modified MoMuLV LTR with myeloproliferative sarcoma virusenhancer, see, e.g., Li et al., J. Neurosci. Methods vol. 189, pp. 56-64(2010) which is incorporated by reference in its entirety for allpurposes), an avian leukemia virus promoter, an Epstein-Barr virusimmediate early promoter, a Rous sarcoma virus promoter, as well ashuman gene promoters such as, but not limited to, the actin promoter,the myosin promoter, the elongation factor-1a promoter, the hemoglobinpromoter, and the creatine kinase promoter. Further, the invention isnot limited to the use of constitutive promoters.

Inducible or repressible promoters are also contemplated for use in thisdisclosure. Examples of inducible promoters include, but are not limitedto a Nuclear Factor of Activated T-cell inducible promoter (NFAT), ametallothionein promoter, a glucocorticoid promoter, a progesteronepromoter, a tetracycline promoter, a c-fos promoter, the T-REx system ofThermoFisher which places expression from the human cytomegalovirusimmediate-early promoter under the control of tetracycline operator(s),and RheoSwitch promoters of Intrexon. Macian et al., Oncogene20:2476-2489 (2001); Karzenowski, D. et al., BioTechiques 39:191-196(2005); Dai, X. et al., Protein Expr. Purif 42:236-245 (2005); Palli, S.R. et al., Eur. J. Biochem. 270:1308-1515 (2003); Dhadialla, T. S. etal., Annual Rev. Entomol. 43:545-569 (1998); Kumar, M. B, et al., J.Biol. Chem. 279:27211-27218 (2004); Verhaegent, M. et al., Annal. Chem.74:4378-4385 (2002); Katalam, A. K., et al., Molecular Therapy 13:S103(2006); and Karzenowski, D. et al., Molecular Therapy 13:S194 (2006),U.S. Pat. Nos. 8,895,306, 8,822,754, 8,748,125, 8,536,354, all of whichare incorporated by reference in their entirety for all purposes.

Expression vectors typically have promoter elements, e.g., enhancers, toregulate the frequency of transcriptional initiation. Typically, theseare located in the region 30-110 bp upstream of the start site, althougha number of promoters have been shown to contain functional elementsdownstream of the start site as well. The spacing between promoterelements frequently is flexible, so that promoter function is preservedwhen elements are inverted or moved relative to one another. In thethymidine kinase (tk) promoter, the spacing between promoter elementscan be increased to 50 bp apart before activity begins to decline.Depending on the promoter, it appears that individual elements canfunction either cooperatively or independently to activatetranscription.

The expression vector may be a bi-cistronic construct or multiplecistronic construct. The two cistrons may be oriented in oppositedirections with the control regions for the cistrons located in betweenthe two cistrons. When the construct has more than two cistrons, thecistrons may be arranged in two groups with the two groups oriented inopposite directions for transcription. Exemplary bicistronic constructsare described in Amendola et al., Nat. Biotechnol. 23:108-116 (2005),which is incorporated by reference in its entirety for all purposes. Thecontrol region for one cistron may be capable of high transcriptionactivity and the other may have low transcriptional activity underconditions of use. One or both control regions may be inducible.Examples of high transcription activity control regions include, forexample, MND, EF1-alpha, PGK1, CMV, ubiquitin C, SV40 early promoter,tetracycline-responsive element promoter, cell-specific promoters, humanbeat-actin promoter, and CBG (chicken beta-globin), optionally includingthe CMV early enhancer. Examples of low transcription activity controlregions include, for example, TRE3G (commercially sold by Clontech, atetracycline-responsive element promoter with mutations that reducebasal expression), T-REx™ (commercially sold by ThermoFisher), and aminimal TATA promoter (Kiran et al., Plant Physiol. 142:364-376 (2006),which is incorporated by reference in its entirety for all purposes),HSP68, and a minimal CMV promoter. Examples of inducible control regionsinclude, for example, NFAT control regions (Macian et al, Oncogene20:2476-2489 (2001)), and the inducible control regions described above.

The bi-cistronic construct may encode a CAR and a polypeptide that is apayload (or makes a payload) to be delivered at a target site. Exemplarypayloads are described above and below. The nucleic acid encoding theCAR can be operably linked to a strong promoter, a weak promoter, and/oran inducible promoter, and optionally, operably linked to a RNA controldevice, DE, RDE, or combination of the foregoing. The CAR can be encodedby nucleic acids in a Side-CAR format. The nucleic acid encoding thepolypeptide can be operably linked to a strong promoter, a weakpromoter, and/or an inducible promoter. The nucleic acid encoding thepolypeptide that is a payload (or makes the payload) can be under thecontrol of an RDE. The RDE may be one that responds to the activationstate of the cell through, for example, glycolytic enzymes such as, forexample, glyceraldehyde phosphate dehydrogenase (GAPDH), enolase (ENO1or ENO3), phosphoglycerate kinase (PGK1), triose phosphate isomerase(TPI1), aldolase A (ALDOA), or phosphoglycerate mutase (PGAM1). The RDEmay also be bound and regulated by other energy metabolism enzymes suchas, for example, transketolase (TKT), malate dehydrogenase (MDH2),succinyl CoA Synthetase (SUGLG1), ATP citrate lyase (ACLY), orisocitrate dehydrogenase (IDH1/2). The host cell can express a CAR thatbinds to its antigen at a target site in a subject. This binding ofantigen at the target site activates the cell causing the cell toincrease glycolysis which induces expression of the nucleic acidencoding the polypeptide under the control of the RDE (bound byglycolytic or other energy metabolism enzymes).

The multicistronic constructs can have three or more cistrons with eachhaving control regions (optionally inducible) and RDEs operably linkedto some or all of the transgenes. These cassettes may be organized intotwo groups that are transcribed in opposite directions on the construct.Two or more transgenes can be transcribed from the same control regionand the two or more transgenes may have IRES (internal ribosome entrysite) sequences operably linked to the downstream transgenes.Alternatively, the two or more transgenes are operably linked togetherby 2A elements as described in Plasmids 101: Multicistronic Vectorsfound at blog.addgene.org/plasmids-101-multicistrnic-vectors. Commonlyused 2A sequences include, for example, EGRGSLLTCGDVEENPGP (T2A) (SEQ IDNO: 24), ATNFSLLKQAGDVEENPGP (P2A) (SEQ ID NO: 25); QCTNYALLKLAGDVESNPGP(E2A) (SEQ ID NO: 26); and VKQTLNFDLLKLAGDVESNPGP (F2A) (SEQ ID NO: 27)all of which can optionally include the sequence GSG at the aminoterminal end. This allows multiple transgenes to be transcribed onto asingle transcript that is regulated by a 3′-UTR with an RDE (or multipleRDEs).

The bicistronic/multicistronic vector can increase the overallexpression of the two or more cistrons (versus introducing the cistronson separate constructs). The bicistronic/multicistronic construct can bederived from a lenti-virus vector. The bicistronic/multicistronicconstruct can encode a CAR and a polypeptide(s) that is encoded on atransgene(s) (e.g., a payload), and the bicistronic construct mayincrease expression of the polypeptide encoded by the transgene(s) whenthe cell is activated by the CAR.

It may be desirable to modify polypeptides described herein. One ofskill will recognize many ways of generating alterations in a givennucleic acid construct to generate variant polypeptides Such well-knownmethods include site-directed mutagenesis, PCR amplification usingdegenerate oligonucleotides, exposure of cells containing the nucleicacid to mutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques (see,e.g., Gillam and Smith, Gene 8:81-97, 1979; Roberts et al., Nature328:731-734, 1987, which is incorporated by reference in its entiretyfor all purposes). The recombinant nucleic acids encoding thepolypeptides of the invention can be modified to provide preferredcodons which enhance translation of the nucleic acid in a selectedorganism.

The polynucleotides can also include polynucleotides includingnucleotide sequences that are substantially equivalent to otherpolynucleotides described herein. Polynucleotides can have at leastabout 80%, more typically at least about 90%, and even more typically atleast about 95%, sequence identity to another polynucleotide. Thenucleic acids also provide the complement of the polynucleotidesincluding a nucleotide sequence that has at least about 80%, moretypically at least about 90%, and even more typically at least about95%, sequence identity to a polynucleotide encoding a polypeptiderecited herein. The polynucleotide can be DNA (genomic, cDNA, amplified,or synthetic) or RNA. Methods and algorithms for obtaining suchpolynucleotides are well known to those of skill in the art and caninclude, for example, methods for determining hybridization conditionswhich can routinely isolate polynucleotides of the desired sequenceidentities.

Nucleic acids which encode protein analogs or variants (i.e., whereinone or more amino acids are designed to differ from the wild typepolypeptide) may be produced using site directed mutagenesis or PCRamplification in which the primer(s) have the desired point mutations.For a detailed description of suitable mutagenesis techniques, seeSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and/or CurrentProtocols in Molecular Biology, Ausubel et al., eds, Green PublishersInc. and Wiley and Sons, N.Y (1994), each of which is incorporated byreference in its entirety for all purposes. Chemical synthesis usingmethods well known in the art, such as that described by Engels et al.,Angew Chem Intl Ed. 28:716-34, 1989 (which is incorporated by referencein its entirety for all purposes), may also be used to prepare suchnucleic acids.

Amino acid “substitutions” for creating variants are preferably theresult of replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, i.e., conservative aminoacid replacements. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

Also disclosed herein are nucleic acids encoding Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CARs. The nucleic acid encoding the Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CAR can be easily prepared from an amino acid sequence ofthe specified CAR combined with the sequence of the RNA control deviceby a conventional method. A base sequence encoding an amino acidsequence can be obtained from the aforementioned NCBI RefSeq IDs oraccession numbers of GenBenk for an amino acid sequence of each element,and the nucleic acid of the present invention can be prepared using astandard molecular biological and/or chemical procedure. For example,based on the base sequence, a nucleic acid can be synthesized, and thenucleic acid of the present invention can be prepared by combining DNAfragments which are obtained from a cDNA library using a polymerasechain reaction (PCR).

The nucleic acids can be linked to another nucleic acid so as to beexpressed under control of a suitable promoter. The nucleic acid can bealso linked to, in order to attain efficient transcription of thenucleic acid, other regulatory elements that cooperate with a promoteror a transcription initiation site, for example, a nucleic acidcomprising an enhancer sequence, a polyA site, or a terminator sequence.In addition to the nucleic acid of the present invention, a gene thatcan be a marker for confirming expression of the nucleic acid (e.g. adrug resistance gene, a gene encoding a reporter enzyme, or a geneencoding a fluorescent protein) may be incorporated.

When the nucleic acid is introduced into a cell ex vivo, the nucleicacid of may be combined with a substance that promotes transference of anucleic acid into a cell, for example, a reagent for introducing anucleic acid such as a liposome or a cationic lipid, in addition to theaforementioned excipients. Alternatively, a vector carrying the nucleicacid of the present invention is also useful. Particularly, acomposition in a form suitable for administration to a living body whichcontains the nucleic acid of the present invention carried by a suitablevector is suitable for in vivo gene therapy.

Introducing Nucleic Acids into Eukaryotic Cells

A process for producing a cell expressing the Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, and/or a transgene operably linked to an RDE(s) includes astep of introducing the nucleic acid encoding a Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, and/or transgene-RDE described herein into a eukaryotic cell.This step can be carried out ex vivo. For example, a cell can betransformed ex vivo with a virus vector or a non-virus vector carryingthe nucleic acid described herein to produce a cell expressing the SmartCAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE.

In a process, a eukaryotic cell as describe above can be used. Theeukaryotic cell can be derived from a mammal, for example, a human cell,or a cell derived from a non-human mammal such as a monkey, a mouse, arat, a pig, a horse, or a dog can be used. The cell used in the processis not particularly limited, and any cell can be used. For example, acell collected, isolated, purified or induced from a body fluid, atissue or an organ such as blood (peripheral blood, umbilical cord bloodetc.) or bone marrow can be used. A peripheral blood mononuclear cell(PBMC), an immune cell, a dendritic cell, a B cell, a hematopoietic stemcell, a macrophage, a monocyte, a NK cell or a hematopoietic cell, anumbilical cord blood mononuclear cell, a fibroblast, a precursoradipocyte, a hepatocyte, a skin keratinocyte, a mesenchymal stem cell,an adipose stem cell, various cancer cell strains, or a neural stem cellcan be used. In the present invention, particularly, use of a T cell, aprecursor cell of a T cell (a hematopoietic stem cell, a lymphocyteprecursor cell etc.) or a cell population containing them is preferable.Examples of the T cell include a CD8-positive T cell, a CD4-positive Tcell, a regulatory T cell, a cytotoxic T cell, and a tumor infiltratinglymphocyte. The cell population containing a T cell and a precursor cellof a T cell includes a PBMC. The aforementioned cells may be collectedfrom a living body, obtained by expansion culture of a cell collectedfrom a living body, or established as a cell strain. Whentransplantation of the produced Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE expressing cell or a cell differentiated from theproduced Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE expressingcell into a living body is desired, it is preferable to introduce thenucleic acid into a cell collected from the living body itself.

The nucleic acid encoding the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE is inserted into a vector, and the vector is introducedinto a cell. The nucleic acid encoding the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE is introduced to the eukaryotic cell bytransfection (e.g., Gorman, et al. Proc. Natl, Acad. Sci. 79.22 (1982):6777-6781, which is incorporated by reference in its entirety for allpurposes), transduction (e.g., Cepko and Pear (2001) Current Protocolsin Molecular Biology unit 9.9; DOI: 10.1002/0471142727.mb0909s36, whichis incorporated by reference in its entirety for all purposes), calciumphosphate transformation (e.g., Kingston, Chen and Okayama (2001)Current Protocols in Molecular Biology Appendix 1C; DOI:10.1002/0471142301.nsa01cs01, which is incorporated by reference in itsentirety for all purposes), cell-penetrating peptides (e.g., Copolovici,Langel, Eriste, and Langel (2014) ACS Nano 2014 8 (3), 1972-1994; DOI:10.1021/nn4057269, which is incorporated by reference in its entiretyfor all purposes), electroporation (e.g Potter (2001) Current Protocolsin Molecular Biology unit 10.15; DOI: 10.1002/0471142735.im1015s03 andKim et al (2014) Genome 1012-19. doi:10.1101/gr.171322.113, Kim et al.2014 describe the Amaza Nucleofector, an optimized electroporationsystem, both of these references are incorporated by reference in theirentirety for all purposes), microinjection (e.g., McNeil (2001) CurrentProtocols in Cell Biology unit 20.1; DOI: 10.1002/0471143030.cb2001s18,which is incorporated by reference in its entirety for all purposes),liposome or cell fusion (e.g., Hawley-Nelson and Ciccarone (2001)Current Protocols in Neuroscience Appendix 1F; DOI:10.1002/0471142301.nsa01fs10, which is incorporated by reference in itsentirety for all purposes), mechanical manipulation (e.g. Sharon et al.(2013) PNAS 2013 110(6); DOI: 10.1073/pnas.1218705110, which isincorporated by reference in its entirety for all purposes or otherwell-known technique for delivery of nucleic acids to eukaryotic cells.Once introduced, Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE nucleic acid can be transiently expressed episomally, orcan be integrated into the genome of the eukaryotic cell using wellknown techniques such as recombination (e.g., Lisby and Rothstein (2015)Cold Spring Harb Perspect Biol. March 2; 7(3). pii: a016535. doi:10.1101/cshperspect.a016535, which is incorporated by reference in itsentirety for all purposes), or non-homologous integration (e.g., Deyleand Russell (2009) Curr Opin Mol Ther. 2009 August; 11(4):442-7, whichis incorporated by reference in its entirety for all purposes). Theefficiency of homologous and non-homologous recombination can befacilitated by genome editing technologies that introduce targeteddouble-stranded breaks (DSB). Examples of DSB-generating technologiesare CRISPR/Cas9, TALEN, Zinc-Finger Nuclease, or equivalent systems(e.g., Cong et al. Science 339.6121 (2013): 819-823, Li et al. Nucl.Acids Res (2011): gkr188, Gaj et al. Trends in Biotechnology 31.7(2013): 39.7-405, all of which are incorporated by reference in theirentirety for all purposes), transposons such as Sleeping Beauty (e.g.,Singh et al (2014) Immunol Rev. 2014 January; 257(1):181-90. doi:10.1111/imr.12137, which is incorporated by reference in its entiretyfor all purposes), targeted recombination using, for example, FLPrecombinase (e.g., O'Gorman, Fox and Wahl Science (1991)15:251(4999):1351-1355, which is incorporated by reference in itsentirety for all purposes), CRE-LOX (e.g., Sauer and Henderson PNAS(1988): 85; 5166-5170), or equivalent systems, or other techniques knownin the art for integrating the nucleic acid encoding the Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side CAR into the eukaryotic cell genome.

The polynucleotide encoding the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE can be integrated into a chromosome of theeukaryotic cell. The polynucleotide encoding the Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, and/or transgene-RDE can be present in the eukaryotic cellextra-chromosomally. The polynucleotide encoding the Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, and/or transgene-RDE can be integrated using a genome editingenzyme (CRISPR, TALEN, Zinc-Finger nuclease), and appropriate nucleicacids (including nucleic acids encoding the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE). The nucleic acid encoding the Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, and/or transgene-RDE can be integrated into the eukaryoticcell chromosome at a genomic safe harbor site, such as, for example, theCCR5, AAVS1, human ROSA26, or PSIP1 loci. (Sadelain et al., Nature Rev.12:51-58 (2012); Fadel et al., J. Virol. 88(17):9704-9717 (2014); Ye etal., PNAS 111(26):9591-9596 (2014), all of which are incorporated byreference in their entirety for all purposes.) The integration of thenucleic acid encoding the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE at the CCR5, PSIP1, or TRAC locus (T-cell receptor αconstant locus) can be done using a gene editing system, such as, forexample, CRISPR, TALEN, Sleeping Beauty Transposase, PiggyBactransposase, or Zinc-Finger nuclease systems. Eyquem et al., Nature543:113-117 (2017), which is incorporated by reference in its entiretyfor all purposes. The eukaryotic cell can be a human, T-lymphocyte and aCRISPR system is used to integrate the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE at the CCR5 or PSIP1 locus. Integration of thenucleic acid at CCR5, PSIP1, or TRAC locus using the CRISPR system alsomay delete a portion, or all, of the CCR5 gene, PSIP1 gene, or TRAClocus. Cas9 in the eukaryotic cell may be derived from a plasmidencoding Cas9, an exogenous mRNA encoding Cas9, or recombinant Cas9polypeptide alone or in a ribonucleoprotein complex. (Kim et al (2014)Genome 1012-19. doi:10.1101/gr.171322.113; Wang et al (2013) Cell 153(4). Elsevier Inc.: 910-18. doi:10.1016/j.cell.2013.04.025, both ofwhich are incorporated by reference in their entirety for all purposes.)

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

Transduction can be done with a virus vector such as a retrovirus vector(including an oncoretrovirus vector, a lentivirus vector, and a pseudotype vector), an adenovirus vector, an adeno-associated virus (AAV)vector, a simian virus vector, a vaccinia virus vector or a sendai virusvector, an Epstein-Barr virus (EBV) vector, and a HSV vector can beused. As the virus vector, a virus vector lacking the replicatingability so as not to self-replicate in an infected cell is preferablyused.

When a retrovirus vector is used to transduce the host cell, the processcan be carried out by selecting a suitable packaging cell based on a LTRsequence and a packaging signal sequence possessed by the vector andpreparing a retrovirus particle using the packaging cell. Examples ofthe packaging cell include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078),GP+E-86 and GP+envAm-12 (U.S. Pat. No. 5,278,056, which is incorporatedby reference in its entirety for all purposes), and Psi-Crip(Proceedings of the National Academy of Sciences of the United States ofAmerica, vol. 85, pp. 6460-6464 (1988), which is incorporated byreference in its entirety for all purposes). A retrovirus particle canalso be prepared using a 293 cell or a T cell having high transfectionefficiency. Many kinds of retrovirus vectors produced based onretroviruses and packaging cells that can be used for packaging of theretrovirus vectors are widely commercially available from manycompanies.

A number of viral based systems have been developed for gene transferinto mammalian cells. A selected gene can be inserted into a vector andpackaged in viral particles using techniques known in the art. Therecombinant virus can then be isolated and delivered to cells of thesubject either in vivo or ex vivo. A number of viral systems are knownin the art. Adenovirus vectors can be used. A number of adenovirusvectors are known in the art and can be used. In addition, lentivirusvectors can be used.

A viral vector derived from a RNA virus can be used to introduce to acell Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE encodingpolynucleotides. The RNA virus vector can encode the reverse complementor antisense strand of the polynucleotide encoding the RNA controldevice and CAR construct (the complementary strand encodes the sensestrand for the RNA control device, DE, RDE, CAR and/or Side-CARconstruct). Thus, the RNA control device should not be active in thesingle stranded, RNA virus vector. The sense strand of the RNA virusconstruct encoding the RNA control device, DE, RDE, CAR, Side-CAR,and/or transgene can be used, and the viral vector with the RNA controldevice, DE, RDE, CAR and/or Side-CAR construct is maintained andreplicated in the presence (or absence) of ligand for the sensor elementof the RNA control device (or under conditions where the RDE is stable)to prevent cleavage of the RNA. The viral vector encoding the sensestrand of the RNA control device, DE, RDE, CAR, Side-CAR, and/ortransgene construct in the viral vector can then be maintained andreplicated with (or without) ligand for the sensor element.

A non-virus vector can be used in combination with a liposome and acondensing agent such as a cationic lipid as described in WO 96/10038,WO 97/18185, WO 97/25329, WO 97/30170 and WO 97/31934 (which areincorporated herein by reference in their entirety for all purposes).The nucleic acid of the present invention can be introduced into a cellby calcium phosphate transduction, DEAE-dextran, electroporation, orparticle bombardment.

Chemical structures with the ability to promote stability and/ortranslation efficiency can be used. The RNA preferably has 5′ and 3′UTRs. The 5′ UTR can be between one and 3000 nucleotides in length. Thelength of 5′ and 3′ UTR sequences to be added to the coding region canbe altered by different methods, including, but not limited to,designing primers for PCR that anneal to different regions of the UTRs.Using this approach, one of ordinary skill in the art can modify the 5′and 3′ UTR lengths required to achieve optimal translation efficiencyfollowing transfection of the transcribed RNA. The 5′ and 3′ UTRs can bethe naturally occurring, endogenous 5′ and 3′ UTRs for the nucleic acidof interest. The UTR sequences that are not endogenous to the nucleicacid of interest can be added by incorporating the UTR sequences intothe forward and reverse primers or by other modification techniquesapplied to the template. The use of UTR sequences that are notendogenous to the nucleic acid of interest can be useful for modifyingthe stability and/or translation efficiency of the RNA. For example, itis known that AU-rich elements in 3′UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

The mRNA may have both a cap on the 5′ end and a 3′ poly(A) tail whichdetermine ribosome binding, initiation of translation and stability mRNAin the cell. On a circular DNA template, for instance, plasmid DNA, RNApolymerase produces a long concatameric product which is not suitablefor expression in eukaryotic cells. The transcription of plasmid DNAlinearized at the end of the 3′ UTR results in normal sized mRNA whichis not effective in eukaryotic transfection even if it is polyadenylatedafter transcription.

In the step of introducing a nucleic acid into a cell, a functionalsubstance for improving the introduction efficiency can also be used(e.g. WO 95/26200 and WO 00/01836, which are incorporated herein byreference in their entirety for all purposes). Examples of the substancefor improving the introduction efficiency include a substance havingability to bind to a virus vector, for example, fibronectin and afibronectin fragment. A fibronectin fragment can have a heparin bindingsite, for example, a fragment commercially available as RetroNetcin(registered trademark, CH-296, manufactured by TAKARA BIO INC.) can beused. Also, polybrene which is a synthetic polycation having an effectof improving the efficiency of infection of a retrovirus into a cell, afibroblast growth factor, V type collagen, polylysine or DEAE-dextrancan be used.

The functional substance can be immobilized on a suitable solid phase,for example, a container used for cell culture (plate, petri dish, flaskor bag) or a carrier (microbeads etc.).

Eukaryotic Cells Expressing the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR

The cell expressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE can be cells in which a nucleic acid encoding a Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE is introduced andexpressed.

Eukaryotic cells can bind to a specific antigen via the CAR, DE-CAR,and/or Side-CAR polypeptide causing the CAR, DE-CAR, and/or Side-CARpolypeptide to transmit a signal into the eukaryotic cell, and as aresult, the eukaryotic cell can be activated. The activation of theeukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR can bevaried depending on the kind of a eukaryotic cell and the intracellularelement of the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR, and can be confirmedbased on, for example, release of a cytokine, improvement of a cellproliferation rate, change in a cell surface molecule, or the like as anindex. For example, release of a cytotoxic cytokine (a tumor necrosisfactor, lymphotoxin, etc.) from the activated cell causes destruction ofa target cell expressing an antigen. In addition, release of a cytokineor change in a cell surface molecule stimulates other immune cells, forexample, a B cell, a dendritic cell, a NK cell, and/or a macrophage.

Eukaryotic cells expressing CAR, Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR constructs can be detected using Protein L (a bacterial surfaceprotein isolated from Peptostreptococcus magnus that selectively bindsto variable light chains (kappa chain) of immunoglobulins. Protein L canbe directly labeled with a reporter (e.g., a light emitting or absorbingmoiety) or can be labeled with an agent such as biotin. When biotin orrelated molecule is used to label the Protein L, binding of Protein L toeukaryotic cells displaying CAR, DE-CAR, and/or Side-CAR polypeptide canbe detected by adding a streptavidin (or similar paired molecule)labeled with reporter (e.g., phycoerythrin). Zheng et al., J.Translational Med., 10:29 (2012), which is incorporated by reference inits entirety for all purposes. Protein L binding to eukaryotic cellscontaining CAR, Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR constructs may show thepresence of antibody light chain, the extracellular domain of a CAR, onthe eukaryotic cell. This method of detecting CAR expression on theeukaryotic cell can also be used to quantitate the amount of CAR,DE-CAR, and/or Side-CAR polypeptide on the surface of the eukaryoticcell. Protein L can be used in QC and QA methodologies for makingeukaryotic cells with the CAR, Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR constructs.

A eukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE can be used as a therapeutic agent to treat adisease. This therapeutic agent can comprise the eukaryotic cellexpressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE as anactive ingredient, and may further comprise a suitable excipient.Examples of the excipient include pharmaceutically acceptable excipientsfor the composition. The disease against which the eukaryotic cellexpressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE isadministered is not particularly limited as long as the disease showssensitivity to the eukaryotic cell. Examples of diseases of theinvention include a cancer (blood cancer (leukemia), solid tumor(ovarian cancer) etc.), an inflammatory disease/autoimmune disease(asthma, eczema), hepatitis, and an infectious disease, the cause ofwhich is a virus such as influenza and HIV, a bacterium, or a fungus,for example, tuberculosis, MRSA, VRE, and deep mycosis. An autoimmunedisease (e.g., pemphigus vulgaris, lupus erythematosus, rheumatoidarthritis) can be treated with a eukaryotic cell expressing a Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE that binds to theimmune proteins that cause the autoimmune disease. For example, theeukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE can target cells that make an antibody which causes theautoimmune disease. The eukaryotic cell expressing the Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE could targetT-lymphocytes which cause the autoimmune disease.

Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE can be used as atherapeutic agent to treat an allergy. Such therapeutic agents cancomprise the eukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE as an active ingredient, and may further comprise asuitable excipient. Examples of the excipient include pharmaceuticallyacceptable excipients for the composition. Examples of allergies thatcan be treated with the eukaryotic cell expressing the Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE include, for example,allergies to pollen, animal dander, peanuts, other nuts, milk products,gluten, eggs, seafood, shellfish, and soy. The eukaryotic cellexpressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE can targetcells that make an antibody which causes the allergic reaction against,for example, pollen, animal dander, peanuts, other nuts, milk products,gluten, eggs, seafood, shellfish, and soy. The targeted cells can be oneor more of B-cells, memory B-cells, plasma cells, pre-B-cells, andprogenitor B-cells. The eukaryotic cell expressing the Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE can targetT-lymphocytes which cause the allergic reaction against, for example,pollen, animal dander, peanuts, other nuts, milk products, gluten, eggs,seafood, shellfish, and soy. The Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE can bind to the idiotypic determinant of theantibody or T-cell receptor.

The eukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE can be administered for treatment of a disease orcondition. These eukaryotic cells can be utilized for prevention of aninfectious disease after bone marrow transplantation or exposure toradiation, donor lymphocyte transfusion for the purpose of remission ofrecurrent leukemia, and the like. The therapeutic agent comprising theeukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE can be an active ingredient and can be administeredintradermally, intramuscularly, subcutaneously, intraperitoneally,intranasally, intraarterially, intravenously, intratumorally, or into anafferent lymph vessel, by parenteral administration, for example, byinjection or infusion, although the administration route is not limited.

The eukaryotic cells with Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE can be characterized prior to administration to thesubject. The eukaryotic cells with Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE can be tested to confirm Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, and/or transgene-RDE expression. The eukaryotic cells withSmart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE can be exposed to alevel of ligand(s) that results in a desired level of CAR, DE-CAR,and/or Side-CAR polypeptide expression in the eukaryotic cell. Thisdesired level of CAR, DE-CAR, and/or Side-CAR polypeptide can produceeukaryotic cells with a desired level of anti-target cell activity,and/or a desired level of proliferative activity when placed in asubject.

The Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE can be used with aT-lymphocyte that has aggressive anti-tumor properties, such as thosedescribed in Pegram et al, CD28z CARs and armored CARs, 2014, Cancer J.20(2):127-133, which is incorporated by reference in its entirety forall purposes. The RNA control device can be used with an armored CAR,DE-CAR, and/or Side-CAR polypeptide in a T-lymphocyte.

The above described Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE embodiments can also include a DE-LEM, RDE-LEM, Smart LEM,Smart-DE-LEM, Smart-RDE-LEM, DE-RDE-LEM, and/or Smart-DE-RDE-LEM toprovide controlled expression of LEM or DE-LEM. The amount of LEM orDE-LEM can be controlled so that its expansion signal is provided at adesired time. This control of the expansion signal is achieved byaltering the amount of ligand (s) for the DE(s) and/or RNA controldevices associated with the LEM or DE-LEM whereby the amount of LEM orDE-LEM is altered. The control of the LEM expansion signal can also beachieved by adding exogenous LEM to the eukaryotic cells at a desiredtime.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise aSmart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE expressing cell, e.g.,a plurality of Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE expressingcells, as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions of the present invention arein one aspect formulated for intravenous administration.

Pharmaceutical compositions may be administered in a manner appropriateto the disease to be treated (or prevented). The quantity and frequencyof administration will be determined by such factors as the condition ofthe patient, and the type and severity of the patient's disease,although appropriate dosages may be determined by clinical trials.

Suitable pharmaceutically acceptable excipients are well known to aperson skilled in the art. Examples of the pharmaceutically acceptableexcipients include phosphate buffered saline (e.g. 0.01 M phosphate,0.138 M NaCl, 0.0027 M KCl, pH 7.4), an aqueous solution containing amineral acid salt such as a hydrochloride, a hydrobromide, a phosphate,or a sulfate, saline, a solution of glycol or ethanol, and a salt of anorganic acid such as an acetate, a propionate, a malonate or a benzoate.An adjuvant such as a wetting agent or an emulsifier, and a pH bufferingagent can also be used. The pharmaceutically acceptable excipientsdescribed in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J.1991) (which is incorporated herein by reference in its entirety for allpurposes) can be appropriately used. The composition can be formulatedinto a known form suitable for parenteral administration, for example,injection or infusion. The composition may comprise formulationadditives such as a suspending agent, a preservative, a stabilizerand/or a dispersant, and a preservation agent for extending a validityterm during storage.

A composition comprising the eukaryotic cells described herein as anactive ingredient can be administered for treatment of, for example, acancer (blood cancer (leukemia), solid tumor (ovarian cancer) etc.), aninflammatory disease/autoimmune disease (pemphigus vulgaris, lupuserythematosus, rheumatoid arthritis, asthma, eczema), hepatitis, and aninfectious disease the cause of which is a virus such as influenza andHIV, a bacterium, or a fungus, for example, a disease such astuberculosis, MRSA, VRE, or deep mycosis, depending on an antigen towhich a CAR, DE-CAR, and/or Side-CAR polypeptide binds.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, intranasally, intraarterially,intratumorally, into an afferent lymph vessel, by intravenous (i.v.)injection, or intraperitoneally. In one aspect, the T cell compositionsof the present invention are administered to a patient by intradermal orsubcutaneous injection. In one aspect, the T-cell compositions of thepresent invention are administered by i.v. injection. The compositionsof T-cells may be injected directly into a tumor, lymph node, or site ofinfection. The administration can be done by adoptive transfer.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). A pharmaceutical composition comprising the eukaryotic cellsdescribed herein may be administered at a dosage of 10⁴ to 10⁹ cells/kgbody weight, in some instances 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. A eukaryotic cellcomposition may also be administered multiple times at these dosages.Eukaryotic cells can also be administered by using infusion techniquesthat are commonly known in immunotherapy (see, e.g., Rosenberg et al.,New Eng. J. of Med. 319:1676, 1988, which is incorporated by referencein its entirety for all purposes).

Uses of Eukaryotic Cells

Nucleic acids encoding Smart CAR(s), DE-CAR(s), RDE-CAR(s),Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s),Side-CAR(s), and/or transgene-RDE(s) can be used to express CAR, DE-CAR,Side-CAR, and/or transgene polypeptides in eukaryotic cells. Theeukaryotic cell can be a mammalian cell, including for example humancells or murine cells. The eukaryotic cells may also be, for example,hematopoietic cells including, e.g., T-cells, natural killer cells,B-cells, or macrophages.

The nucleic acids encoding the Smart CAR(s), DE-CAR(s), RDE-CAR(s),Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s),Side-CAR(s), and/or transgene-RDE(s) can be used to express a desiredlevel of CAR, DE-CAR, and/or Side-CAR polypeptide on the surface of theeukaryotic cell. In this aspect, the DE, RNA control device, RDE, and/orSide-CAR controls the level of CAR, DE-CAR, and/or Side-CAR polypeptideexpression, at least in part, and by modulating the level of activity ofthe DE, RNA control device, RDE, and/or Side-CAR, a desired amount ofCAR, DE-CAR, and/or Side-CAR polypeptide is expressed and displayed onthe surface of the eukaryotic cell. The amount of CAR, DE-CAR, and/orSide-CAR polypeptide can be measured using antibodies specific for theCAR, DE-CAR, and/or Side-CAR polypeptide. The amount of CAR, DE-CAR,and/or Side-CAR polypeptide can be measured using the antigen recognizedby the extracellular element. The amount of CAR, DE-CAR, and/or Side-CARpolypeptide can be measured in a functional assay of target cellkilling. The amount of CAR, DE-CAR, and/or Side-CAR polypeptide can bemeasured in a functional assay for eukaryotic cell proliferation(induced by the CAR, DE-CAR, and/or Side-CAR polypeptide). The aboveeukaryotic cell can be a T-lymphocyte or a natural killer cell or amacrophage or other phagocytic cell type.

The ligand for the DE, the ligand for the RNA control device sensor,and/or the ligand for the Side-CAR can be added in increasing amountsuntil a desired level of eukaryotic cell activity is obtained. Thedesired eukaryotic cell activity can be killing of a target cell. Targetcell killing can occur over a desired time period, e.g., the killing ofa certain number of target cells in 12 hours, or 24 hours, or 36 hours,or two days, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, or two months, or 3, 4,5, or 6 months. Target cell killing can be expressed as a half-life fora standardized number of target cells. The half-life of target cellkilling can be 12 hours, 24 hours, 36 hours, or 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,or 28 days, or two months, or 3, 4, 5, or 6 months. The desiredeukaryotic cell activity can be proliferation. The cell proliferationcan occur with a doubling time of 12 hours, 24 hours, 36 hours, twodays, or 3, 4, 5, 6, or 7 days. The above eukaryotic cell can be aT-lymphocyte or a natural killer cell or a macrophage or otherphagocytic cell type.

A regime of different amounts of ligand (for the sensor, DE, and/orSide-CAR) can be added over time so that different desired levels ofCAR, DE-CAR, and/or Side-CAR polypeptide are present on the eukaryoticcell at different times. For example, in the treatment of cancer inpatients with Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR T-cellsor Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR natural killercells, the amount of CAR, DE-CAR, and/or Side-CAR polypeptide expressionmay be reduced initially to reduce toxicity from tumor lysis, and astumor mass is cleared, the amount of CAR, DE-CAR, and/or Side-CARpolypeptide expression can be increased to kill the remaining tumorcells as these become more rare within the body. The CAR, DE-CAR, and/orSide-CAR polypeptide expression may be increased initially, and as tumormass is reduced the CAR, DE-CAR, and/or Side-CAR polypeptide expressionlevel is reduced to reduce killing of healthy tissue that also mayexpress target antigen. The reactivity towards tumor cells can bemodulated so that the ratio of tumor cell killing to killing of normaltissue is maintained within a desired range. The amount of CAR, DE-CAR,and/or Side-CAR polypeptide expressed on the surface of the eukaryoticcell can be reduced by eukaryotic cell proliferation. As the eukaryoticcells proliferate, CAR, DE-CAR, and/or Side-CAR polypeptide will bediluted if the expression level from the Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR nucleic acid is insufficient to keep theCAR, DE-CAR, and/or Side-CAR polypeptide copy number at the level foundin the parent eukaryotic cell (i.e., if the parent cell does not doubleits amount of CAR, DE-CAR, and/or Side-CAR polypeptide then eachdaughter cell will have a decreased amount CAR, DE-CAR, and/or Side-CARpolypeptide compared to the parent cell). The CAR, DE-CAR, and/orSide-CAR polypeptide can be designed to have a short half-life, incomparison to the doubling time for the eukaryotic cell in the subject.The ligand(s) can have a short half-life in the subject when compared tothe doubling time of the eukaryotic cell in the subject. An anti-ligandantibody or a different ligand binding molecule can be administered tothe subject or given to the eukaryotic cells (in vitro) so that theligand binds to the antibody or ligand binding molecule and cannot reactwith the DE, RNA control device sensor, and/or Side-CAR. The aboveeukaryotic cell can be a T-lymphocyte or a natural killer cell or amacrophage or other phagocytic cell type.

Eukaryotic cells with Smart CAR(s), DE-CAR(s), RDE-CAR(s),Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s)and/or Side-CAR(s) can express a desired amount of CAR, DE-CAR, and/orSide-CAR polypeptide so that a subject containing the eukaryotic cellswith the CAR, DE-CAR, and/or Side-CAR polypeptide can produce atherapeutic level of target cell killing while keeping toxicity andadverse events at acceptable levels. The above eukaryotic cell can be aT-lymphocyte or a natural killer cell or a macrophage or other immunecell type. For example, Smart CAR(s), DE-CAR(s), RDE-CAR(s),Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s)and/or Side-CAR(s) of the invention can be used to reduce tumor lysissyndrome, cytokine storms, or healthy tissue killing by T-lymphocyteswith Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR.

The Smart CAR, Smart-DE-CAR, Smart-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR can be associated with two or more RNA control devices.Different amounts of the two or more ligands for the DE, Side-CAR,and/or two or more RNA control devices can be added to the eukaryoticcells to produce a desired amount of CAR, DE-CAR, and/or Side-CARpolypeptide in the eukaryotic cell. Different regimes of combinations ofthe ligands can be applied to the eukaryotic cells to produce a desiredprofile over time of the amount of CAR, DE-CAR, and/or Side-CARpolypeptide on the surface of the eukaryotic cell.

The desired amount of CAR expression can activate effector function in aT lymphocyte or natural killer cell with a minimal amount of Tlymphocyte exhaustion, dysfunction, and/or other dysregulatory process(such as change of cell fate, or change in metabolism effecting forexample glycolysis). The desired amount of CAR expression can activateeffector function in a T lymphocyte or natural killer cell with aminimal amount of inhibition, exhaustion, and/or dysfunction of the cellwhich causes the T lymphocyte or natural killer cell to become exhaustedor dysfunctional. The desired amount of CAR expression can provide adesired dynamic range of effector function in response to target antigenpresent at a target cell. The desired amount of CAR expression canprovide a desired range of effector function. The desired amount of CARexpression can provide a small dynamic range of effector function (actslike an on-off switch), for example, when target cells have a highdensity of target antigen and healthy, nontarget cells have a lowdensity of target antigen. The desired amount of CAR expression canallow the eukaryotic cell to distinguish between target cells (on—CARsactivate effector function) versus healthy, normal cells (off—CARactivates little or no effector function). The desired amount of CARexpression can increase the avidity of the engineered eukaryotic cell(e.g., T lymphocytes or natural killer cells) for target cells wherebyeffect on target cells is increased. The desired amount of CARexpression can increase the avidity of the engineered eukaryotic cell(e.g., T lymphocytes or natural killer cells) for target cells wherebytarget cell killing is enhanced. The desired amount of CAR expressioncan increase the avidity of the engineered hematopoietic cell (e.g., Tlymphocytes or natural killer cells) for target cells whereby cytokinesecretion is enhanced.

T-cells (e.g., CD4+ or CD8+) or natural killer cells can be engineeredwith a polynucleotide encoding a Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR. Ligand for the RNA control device, DE, or Side CAR is added tothe T-cells (e.g., CD4+ or CD8+) or natural killer cells can be added inincreasing amounts to obtain a desired amount of effector function. Thedesired amount of effector function can be an optimized amount ofeffector function with a known amount (and/or density) of target antigenon target cells. Effector function can be target cell killing,activation of host immune cells, cytokine secretion, production ofgranzymes, production of apoptosis inducing ligands, production of otherligands that modulate the immune system, etc. The effector function canbe secretion of cytokines such as, for example, IL-2, IFN-γ, TNF-α,TGF-β, and/or IL-10. Effector function can be the killing of targetcells. Target cells can be killed with granzymes. Target cells can beinduced to undergo apoptosis. Eukaryotic cells with CARs can kill targetcells through apoptosis and granzymes. The optimal concentration forligand may increase effector function. The optimal concentration ofligand can increase the dynamic range of effector function (effectoractivity in response to target antigen). The optimal concentration forligand may provide a desired amount of effector function and a desired(or tolerable) amount of exhaustion, dysfunction, and/or inhibition ofthe eukaryotic cell (e.g., T-lymphocyte or natural killer cell). Theoptimal amount of activity can yield a desired proliferative activity.The optimal amount of activity can be an amount of target binding or anamount of an effector activity (e.g., target cell killing). The optimalCAR, DE-CAR and/or Side-CAR expression can provide a desired rate ofmemory cell formation when the eukaryotic cell is an appropriate immunecell. Other eukaryotic cell activities that may be optimized include anyactivities useful in the treatment of disease, including, for example,rate of memory cell formation, release rate of cytokines, phagocytosis,binding of target, recruitment of innate myeloid or lymphoid cells,epitope spreading, development of exhaustion, development of celldysfunction, and/or inhibition of eukaryotic cell function.

The RDE, DE, RNA control device, or Side CAR regulatory element can beused to control expression of a transgene. This transgene expression candeliver a payload at a target site. Expression of the transgene cancause a desired change in the eukaryotic cell. An RDE regulated by GAPDHcan be used for payload delivery, and the eukaryotic cell (e.g., T-cell,natural killer cell, B-cell, macrophage, dendritic cell, or otherantigen presenting cell) can be activated (e.g., by a CAR) when itreaches the target site. Upon activation of the eukaryotic cell at thetarget site through the CAR, the cell induces glycolysis and the GAPDHreleases from the RDE allowed payload expression and delivery. Thetarget site can be a tumor or infection and the transgene could encode acytokine, a chemokine, an antibody, a checkpoint inhibitor, a granzyme,an apoptosis inducer, complement, an enzyme for making a cytotoxic smallmolecule, an enzyme that cleaves peptides or saccharides (e.g., fordigesting a biofilm), other cytotoxic compounds, or other polypeptidesthat can have a desired effect at the target site. Checkpoint inhibitorsinclude agents that act at immune checkpoints including, for example,cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), programmed celldeath protein (PD-1), Killer-cell Immunoglobulin-like Receptors (KIR),and Lymphocyte Activation Gene-3 (LAG3). Examples of checkpointinhibitors that may be used as payloads include, for example, Nivolumab(Opdivo), Pembrolizumab (Keytruda), Atezolizumab (Tecentriq), Ipilimumab(Yervoy), Lirilumab, and BMS-986016. Nivolumab, Atezolizumab andPembrolizumab act at the checkpoint protein PD-1 and inhibit apoptosisof anti-tumor immune cells. Some checkpoint inhibitors prevent theinteraction between PD-1 and its ligand PD-L1. Ipilimumab acts at CTLA4and prevents CTLA4 from downregulating activated T-cells in the tumor.Lirilumab acts at KIR and facilitates activation of Natural Killercells. BMS-986016 acts at LAG3 and activates antigen-specificT-lymphocytes and enhances cytotoxic T cell-mediated lysis of tumorcells. Cytokines can include, for example, IL-2, IL-12, IL-15, IL-18,IFN-γ, TNF-α, TGF-β, and/or IL-10. Cytotoxic agents can include, forexample, granzymes, apoptosis inducers, complement, or a cytotoxic smallmolecule. The payload delivered at a target site (e.g., non-tumor targetsite) can be a factor that protects the target site such as, forexample, an anti-inflammatory, a factor that attracts T-regulatory cellsto the site, or cytokines or other factors that cause suppression andreduction in immune activity. The payload can be an enzyme that cleavespeptides or saccharides, for example hyaluronidase, heparanase,metalloproteinases and other proteinases which can be used, for example,to digest an undesired biofilm. The payload can be an imaging agent thatallows the target site to be imaged. The payload may be a polypeptidethat can be imaged directly, or it can be a polypeptide that interactswith a substrate to make a product that can be imaged, imagingpolypeptides include, for example, thymidine kinase (PET), dopamine D2(D2R) receptor, sodium iodide transporter (NIS), dexoycytidine kinase,somatostatin receptor subtype 2, norepinephrine transporter (NET),cannabinoid receptor, glucose transporter (Glut1), tyrosinase, sodiumiodide transporter, dopamine D2 (D2R) receptor, modified haloalkanedehalogenase, tyrosinase, β-galactosidase, and somatostatin receptor 2.These reporter payloads can be imaged using, for example, opticalimaging, ultrasound imaging, computed tomography imaging, opticalcoherence tomography imaging, radiography imaging, nuclear medicalimaging, positron emission tomography imaging, tomography imaging, photoacoustic tomography imaging, x-ray imaging, thermal imaging, fluoroscopyimaging, bioluminescent imaging, and fluorescent imaging. These imagingmethods include Positron Emission Tomography (PET) or Single PhotonEmission Computed Tomography (SPECT).

Thymidine kinase can be used with PET reporter probes such as, forexample, [¹⁸F]9-(4-[¹⁸F]-fluoro-3-hydroxymethylbutyl)-guanine, afluorine-18-labelled penciclovir analogue, which when phosphorylated bythymidine kinase (TK) becomes retained intracellularly, or is 5-(76)Br-bromo-2′-fluoro-2′-deoxyuridine. The relevant reporter probes foreach of the PET reporters are well known to the skilled artisan. Anexemplary reporter probe for dopamine D2 (D2R) receptor is3-(2′-[¹⁸F]fluoroethyl)spiperone (FESP) (MacLaren et al., Gene Ther.6(5):785-91 (1999)). An exemplary reporter probe for the sodium iodidetransporter is ¹²⁴I, which is retained in cells following transport bythe transporter. An exemplary reporter probe for deoxycytidine kinase is2′-deoxy-2′-¹⁸F-5-ethyl-1-β-d-arabinofuranosyluracil (¹⁸F-FEAU). Anexemplary reporter probe for somatostatin receptor subtype 2 is ¹¹¹In-,^(99m/94m)Tc-, ⁹⁰Y-, or ¹⁷⁷Lu-labeled octreotide analogues, for example⁹⁰Y-, or ¹⁷⁷Lu-labeled DOTATOC (Zhang et al., J Nucl Med. 50(suppl2):323 (2009)); ⁶⁸Ga-DOTATATE; and ¹⁷⁷In-DOTABASS (see. e.g., Brader etal., J Nucl Med. 54(2):167-172 (2013), incorporated herein byreference). An exemplary reporter probe for norepinephrine transporteris ¹¹C-m-hydroxyephedrine (Buursma et al., J Nucl Med. 46:2068-2075(2005)). An exemplary reporter probe for the cannabinoid receptor is¹¹C-labeled CB2 ligand, ¹¹C-GW405833 (Vandeputte et al., J Nucl Med.52(7):1102-1109 (2011)). An exemplary reporter probe for the glucosetransporter is [¹⁸F]fluoro-2-deoxy-d-glucose (Herschman, H. R., Crit RevOncology/Hematology 51:191-204 (2004)). An exemplary reporter probe fortyrosinase is N-(2-(diethylamino)ethyl)-¹⁸F-5-fluoropicolinamide (Qin etal., Sci Rep. 3:1490 (2013)). Other reporter probes are described in theart, for example, in Yaghoubi et al., Theranostics 2(4):374-391 (2012),incorporated herein by reference.

An exemplary photoacoustic reporter probe for β-galactosidase is 5-bromochloro-3-indolyl-β-D-galactoside (X-gal) (Li et al., J Biomed Opt.12(2):020504 (2007)). Exemplary X-ray reporter includes, among others,somatostatin receptor 2, or other types of receptor based bindingagents. The reporter probe can have a radiopaque label moiety that isbound to the reporter probe and imaged, for example, by X-ray orcomputer tomography. Exemplary radiopaque label is iodine, particularlya polyiodinated chemical group (see, e.g., U.S. Pat. No. 5,141,739), andparamagnetic labels (e.g., gadolinium), which can be attached to thereporter probe by conventional means. Optical imaging agents include,for example, a fluorescent polypeptide. Fluorescent polypeptidesinclude, for example, green fluorescent protein from Aequorea victoriaor Renilla reniformis, and active variants thereof (e.g., bluefluorescent protein, yellow fluorescent protein, cyan fluorescentprotein, etc.); fluorescent proteins from Hydroid jellyfishes, Copepod,Ctenophora, Anthrozoas, and Entacmaea quadricolor, and active variantsthereof; and phycobiliproteins and active variants thereof. The opticalimaging agent can also be a bioluminescent polypeptide. These include,for example, aequorin (and other Ca⁺² regulated photoproteins),luciferase based on luciferin substrate, luciferase based onCoelenterazine substrate (e.g., Renilla, Gaussia, and Metridina), andluciferase from Cypridina, and active variants thereof.

The desired amount of CAR expression may consider the target cellconcentration, density of target antigen, whether target cells areassociated with other target cells (e.g., in a tumor or a biofilm), thebinding affinity (K_(d)) of the extracellular element (antigen bindingelement) for the target antigen, and the concentration of eukaryoticcells with CARs. These parameters can be used to arrive at a desireddensity of CARs on the eukaryotic cell which will define the desiredlevel of CAR expression. The desired amount of CAR expression may alsoconsider the amount of inhibitory receptors (IR) expressed on theeukaryotic cell, and the amount of inhibitory receptor ligand (IRL)expressed on target (and other) cells. The following equations can beused, at least in part, to arrive at the desired amount of CARexpression:

$\begin{matrix}\begin{matrix}{{{CAR}\mspace{14mu}{Expression}} = {{{\left\lbrack {{target}\mspace{14mu}{cell}} \right\rbrack\left\lbrack {{target}\mspace{14mu}{antigen}\mspace{14mu}{density}} \right\rbrack}\left\lbrack K_{d} \right\rbrack}\quad}} \\\left\lbrack {{host}\mspace{14mu}{cells}} \right\rbrack\end{matrix} & I \\\frac{\begin{matrix}{{{CAR}\mspace{14mu}{Expression}} = {{\left\lbrack {{target}\mspace{14mu}{cell}} \right\rbrack\left\lbrack {{target}\mspace{14mu}{antigen}\mspace{14mu}{density}} \right\rbrack}\left\lbrack K_{d} \right\rbrack}} \\\left. {\left\lbrack \quad \right.{host}\mspace{14mu}{cells}} \right\rbrack\end{matrix}}{\lbrack{IR}\rbrack\lbrack{IRL}\rbrack} & {II}\end{matrix}$

Equation II can optionally include [target antigen density on healthycells] in the denominator.

The desired amount of CAR expression can produce a desired number ofCARs on the surface of the eukaryotic cell. The desired amount of CARexpression can produce 2-100,000 CARs (or DE-CARs or Side-CARs) on thesurface of the eukaryotic cell. The eukaryotic cell can be aT-lymphocyte and the number of CARs (or DE-CARs or Side-CARs) on thesurface of the T-lymphocyte can be 2-100,000. The CAR, DE-CAR, and/orSide-CAR can bind to target ligand with an affinity in the micromolar(μM) range and the desired number of CARs, DE-CARs, and/or Side-CARs onthe surface of the T-lymphocyte or natural killer cell can be100-500,000. The CAR, DE-CAR, and/or Side-CAR can bind to target ligandwith an affinity in the nanomolar (nM) range and the desired number ofCARs, DE-CARs, and/or Side-CARs on the surface of the T-lymphocyte ornatural killer cell can be 2-100,000.

The desired amount of CAR expression can produce 2-1,000, 10-1,000,10-5,000, 10-10,000, 10-50,000, 10-100,000, 10-500,000, or 10-1,000,000CARs (or DE-CARs or Side-CARs) on the surface of the eukaryotic cell.The desired amount of CAR expression can produce 100-1,000, 100-5,000,100-10,000, 100-50,000, 100-100,000, 100-500,000, 100-1,000,000,1,000-5,000, 1,000-10,000, 1,000-50,000, 1,000-100,000, 1,000-500,000,1,000-1,000,000, 10,000-50,000, 10,000-100,000, 10,000-500,000, or10,000-1,000,000 CARs (or DE-CARs or Side-CARs) on the surface of theeukaryotic cell. The desired amount of CAR expression can produce atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,260, 280, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 2000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000,10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000,19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000,28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000,37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000,46,000, 47,000, 48,000, 49,000, 50,000, 51,000, 52,000, 53,000, 54,000,55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000or 100,000 CARs (or DE-CARs or Side-CARs) on the surface of theeukaryotic cell. The desired amount of CAR expression can produce fewerthan 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,260, 280, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 2000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000,10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000,19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000,28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000,37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000,46,000, 47,000, 48,000, 49,000, 50,000, 51,000, 52,000, 53,000, 54,000,55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000or 100,000 CARs (or DE-CARs or Side-CARs) on the surface of theeukaryotic cell. The desired amount of CAR expression can produce 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 2000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000,11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000,20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000,29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000,38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000,47,000, 48,000, 49,000, 50,000, 51,000, 52,000, 53,000, 54,000, 55,000,60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000 or100,000 CARs (or DE-CARs or Side-CARs) on the surface of the eukaryoticcell. The eukaryotic cell can be a T-lymphocyte and the number of CARs(or DE-CARs or Side-CARs) on the surface of the T-lymphocyte can be100-100,000. The CAR, DE-CAR, and/or Side-CAR can bind to target ligandwith an affinity in the micromolar (μM) range (e.g., 1-500 μM) and thedesired number of CARs, DE-CARs, and/or Side-CARs on the surface of theT-lymphocyte or natural killer cell can be 100-100,000. The CAR, DE-CAR,and/or Side-CAR can bind to target ligand with an affinity in themicromolar (μM) range and the desired number of CARs, DE-CARs, and/orSide-CARs on the surface of the T-lymphocyte or natural killer cell canbe 100-1,000, 100-5,000, 100-10,000, 100-50,000, 100-100,000,100-500,000, 100-1,000,000, 1,000-5,000, 1,000-10,000, 1,000-50,000,1,000-100,000, 1,000-500,000, 1,000-1,000,000, 10,000-50,000,10,000-100,000, 10,000-500,000, or 10,000-1,000,000. The CAR, DE-CAR,and/or Side-CAR can bind to target ligand with an affinity in thenanomolar (nM) range and the desired number of CARs, DE-CARs, and/orSide-CARs on the surface of the T-lymphocyte or natural killer cell canbe 10-100,000. The CAR, DE-CAR, and/or Side-CAR can bind to targetligand with an affinity in the nanomolar (nM) range (e.g., 1-500 nM) andthe desired number of CARs, DE-CARs, and/or Side-CARs on the surface ofthe T-lymphocyte or natural killer cell can be 100-1,000, 100-5,000,100-10,000, 100-50,000, 100-100,000, 100-500,000, 100-1,000,000,1,000-5,000, 1,000-10,000, 1,000-50,000, 1,000-100,000, 1,000-500,000,1,000-1,000,000, 10,000-50,000, 10,000-100,000, 10,000-500,000, or

The desired amount of CAR expression can be an amount which gives adesired amount of area under the curve for a desired activity (e.g.,target cell killing and/or cytokine release as a function of time). Thedesired amount of CAR expression can be the amount which gives themaximal amount of area under the curve for a desired activity (e.g.,target cell killing and/or cytokine release). The desired amount of CARexpression can be the amount which gives the optimal amount of areaunder the curve for a desired activity (e.g., target cell killing and/orcytokine release). The desired amount of CAR expression can be theamount which gives the desired activity rate maximum (analogous toC_(max)) for a desired activity (e.g., target cell killing and/orcytokine release). The desired amount of CAR expression can be theamount which gives the maximal activity rate for a desired activity(e.g., target cell killing and/or cytokine release). The desired amountof CAR expression can be the amount which gives the optimal activityrate for a desired activity (e.g., target cell killing and/or cytokinerelease). Eukaryotic cell activities that may be customized include, forexample, any activities useful in the treatment of disease, including,for example, proliferation rate, rate of memory cell formation, releaserate of cytokines, phagocytosis, binding of target, recruitment ofinnate myeloid or lymphoid cells, epitope spreading, development ofexhaustion, development of cell dysfunction, and/or inhibition ofeukaryotic cell function. In an aspect, the desired amount of CAR,DE-CAR, and/or Side-CAR polypeptide(s) can be such so that exhaustion,dysfunction and/or inhibitory signals for the eukaryotic cell can beminimized.

The desired amount of CAR expression can be the amount which gives thedesired amount of area under the curve for a desired activity (e.g.,target cell killing and/or cytokine release) with a desired amount ofexhaustion, dysfunction, and/or inhibitory receptor activity and/orexpression. The desired amount of CAR expression can be the amount whichgives the desired amount of area under the curve for a desired activity(e.g., target cell killing and/or cytokine release) with a tolerableamount of inhibitory activity, T-lymphocyte dysfunctional activity,and/or exhaustion. The desired amount of CAR expression can be theamount which gives the maximal amount of area under the curve for adesired activity (e.g., target cell killing and/or cytokine release)with a minimal amount of host cell dysfunction, exhaustion, and/orinhibitory receptor expression. The desired amount of CAR expression canbe the amount which gives the maximal amount of area under the curve fora desired activity (e.g., target cell killing and/or cytokine release)with a minimal amount of host cell dysfunction. The desired amount ofCAR expression can be the amount which gives the maximal amount of areaunder the curve for a desired activity (e.g., target cell killing and/orcytokine release) with a desired ratio of area under the curve for thedesired activity versus area under the curve for an inhibitory activity.The desired amount of CAR expression can be the amount which gives themaximal amount of area under the curve for a desired activity (e.g.,target cell killing and/or cytokine release) with a desired ratio ofarea under the curve for the desired activity versus area under thecurve for host cell dysfunction. The desired amount of CAR expressioncan be the amount which gives a maximal ratio of area under the curvefor effector function versus area under the curve for inhibitoryreceptor activity. The desired amount of CAR expression can be theamount which gives a maximal ratio of area under the curve for effectorfunction versus area under the curve for host cell dysfunction activity.The desired amount of CAR expression can be the amount which gives amaximal ratio of area under the curve for effector function versus areaunder the curve for inhibitory or dysfunction activity, and/orexhaustion.

The desired amount of CAR expression can be the amount that gives adesired area under the curve for a desired activity over a period oftime, and a desired length of time for the CAR cells to recover (ormaintain) the desired activity (during an off period) for the next oncycle (cycle for effector function). The desired amount of CARexpression can be the amount that gives a desired area under the curvefor a desired activity, and a desired length of time for the CAR cellsto regain the desired effector function (during an off period). Thedesired amount of CAR expression can be correlated with the rate atwhich effector function is induced, the rate at which inhibitoryfunction, exhaustion, and/or dysfunction of the T-lymphocyte (or naturalkiller cell) is induced, and the rate at which inhibitory receptors arelost and/or inhibitory activity is lost from the T-lymphocyte or naturalkiller cell when CAR expression is turned off.

The desired amount of CAR expression can be cycled so the eukaryoticcell goes through periods of on (activation of eukaryotic cell andeffector function) and off (no activation—down regulation of inhibitorreceptors). The desired amount of CAR expression can be cycled so thatcells activated by the CAR eukaryotic cell are cycled, e.g., cellsactivated by cytokines expressed by the eukaryotic cell with the CAR(and other immune cells that are activated by the eukaryotic cell). TheT lymphocytes and/or natural killer cells can be engineered to knock outinhibitory receptor expression, including for example, PD-1 and CTLA-4.Cycling of CAR expression along with activated and deactivated statescan be done on a population basis. Cycling of CAR expression along withactivated and deactivated states can be done for individual eukaryoticcells.

Optimal expression of a CAR, DE-CAR, or Side-CAR can be determined bytaking the first derivative (to find the maximum) of a desired effectoractivity as a function of time. The following equations can be used todescribe the effector activity, which can be dependent on the rate ofgeneration of activation related signaling molecules (F_(A)), subtractedby the rate of generation of inhibition related signaling molecules(F_(I)), and optionally subtracted by some loss of effector functionparameter Q_(m) that is dependent on alterations in metabolic state.

$\frac{dE}{dt} = {F_{A} - F_{I} - Q_{m}}$

F_(A) is the rate of generation of activation molecules which can alsobe expressed as dN_(A)/dt. F_(A) can be linearly dependent on N_(RT)(the instantaneous number of receptors bound to targets), as well as aconstant, c_(activation).

$F_{A} = {\frac{{dN}_{A}}{dt} = {N_{RT} \cdot c_{activation}}}$

N_(RT) can be found through the reaction equation and the correspondingrates of association and dissociation.

${\lbrack R\rbrack\lbrack T\rbrack}\overset{k\; 1}{\rightarrow}{{\lbrack{RT}\rbrack\lbrack{RT}\rbrack}\overset{{k\_}1}{\rightarrow}{\lbrack R\rbrack\lbrack T\rbrack}}$$\frac{dN_{RT}}{dt} = {{k1N_{R}N_{T}} - {{k\_}1N_{RT}}}$

Solving this first order ODE for dN_(RT)N _(RT) =e ^((k1N) ^(R) ^(N) ^(T) ^(−k_1)t)

At equilibrium conditions dN_(RT)/dt can be set to 0, leaving N_(RT)which can be substituted into the equation for F_(A), purely dependenton NR (the number of receptors), NT (number of targets), k1 and k_1which are the rate constants of association and dissociation of thereceptor target complex, which is defined by antibody affinity for itstarget.

$N_{RT} = \frac{k1N_{R}N_{T}}{{k\_}1}$

For F_(I), we seek to provide a negative feedback for activation, whichis consistent with observed biological function. Here we express it asan exponential function of N_(A) and a constant c₂ and c_(inhibition).

$F_{I} = {\frac{{dN}_{I}}{dt}{e^{C_{2}N_{A}} \cdot c_{inhibition}}}$

The optional form of Q can be an arbitrary function of N_(A) which isoften dependent on the activation state as well.Q=f(N _(A))·c _(metabolism)

These equations can be used to plot for E, and for a given nT, k1, k_1and varying N_(R), the maximum E can be identified as depicted in FIG. 4. FIG. 4 shows graphs of effector function (E), activation (of effectorfunction) signal or molecules (NA), and inhibition (of effectorfunction) signal or molecules (NI) as a function of the number of CARreceptors on the cell. The graphs in FIG. 4 have a set targetconcentration and the graphs show that maximal (and in some caseoptimal) effector function occurs at CAR receptor number less than themaximal number of CAR receptors that can be expressed on the eukaryoticcell surface.

The desired amount of CAR expression can consider the competingprocesses of eukaryotic host cell activation (as correlated with CARexpression) and inhibition of the effector function of the host cell(deactivation, dysfunction, and/or exhaustion). The desired amount ofCAR expression can consider the competing processes of T-lymphocyte ornatural killer cell activation (as correlated with CAR expression) andinhibition of effector function (deactivation, dysfunction and/orexhaustion). FIG. 5 shows that T-lymphocyte activation (effectorfunction of the host cell) decreases as the inhibition (exhaustion,dysfunction and/or deactivation) rises. The operable window for theSmart-CAR host cell can be defined by these two processes as shown inFIG. 5 . The Smart-CAR host cells can be exposed to cycles of ligandthat keep the effector function in the window of operation (by turningoff CAR expression the host cells should reduce activation which in turnwill reduce expression of inhibitory process, e.g., inhibitoryreceptors, in the host cell).

FIG. 7 shows calculated graphs for effector function (E), number oftarget cells (nT), number of CAR receptors (nR), and number of CARreceptor-target cell interactions (nRT) versus time as calculated by theprogram in python code at the end of this specification. Maximaleffector function (E) can be reached at time 0.7 despite the fact thatthe CAR receptors are engaged with antigen throughout the time of thesimulation. The python program at the end of the specification can beused with different parameters of number of target cells (nT), number ofCAR receptors (nR), and number of CAR receptor-target cell interactions(nRT) to model maximal effector function.

FIG. 7 shows a chart looking at effector activity as a function of CARcopy number, number of target antigens (epitopes), and CAR bindingaffinity for its antigen (epitope). Optimal CAR activity can be obtainedwhen the variables of CAR copy number, CAR affinity for antigen, andcopy number of target epitopes are coordinated to produce a desiredamount of effector activity in the cell. The control devices can controlCAR copy number to maintain the amount of effector function in theoptimal CAR activity range of FIG. 7 . As the copy number of targetepitope decreases (as target cells are killed) the CAR copy number maybe increased to compensate for the reduced amount of target antigenavailable to bind to the CARs and activate them.

The desired amount of CAR expression can be cycled so the eukaryoticcell (or population of eukaryotic cells) goes through periods of on(activation of cell and effector function) and off (no activation—downregulation of inhibitor receptors, inhibitor activity, and/ordysfunction). The desired amount of CAR expression can be cycled so theactivity of cells induced by the CAR eukaryotic cell is cycled, e.g.,cells activated by cytokines expressed by the eukaryotic cell with theCAR are cycled (and other immune cells that are activated by theeukaryotic cell). The T lymphocytes and/or natural killer cells can beengineered to knock out inhibitory receptor expression or knock outother factors that cause dysfunction and/or exhaustion, including forexample, PD-1, CTLA-4, TIM-3, LAG-3, T-bet and/or Blimp-1. Cycling ofCAR expression along with activated and deactivated states can be doneon a population basis. Cycling of CAR expression along with activatedand deactivated states can be done for individual eukaryotic cells.

Smart CAR(s), DE-CAR(s), RDE-CAR(s), Smart-RDE-CAR(s), DE-RDE-CAR(s),Smart-DE-CAR(s), Smart-DE-RDE-CAR(s) and/or Side-CAR(s) can be used forgenetically engineering T-cells for cancer immunotherapy. When used forsome immunotherapy applications, leukocytes are removed from a patientthrough leukopheresis and T-lymphocytes are preferentially sorted andsaved. T-lymphocytes are subjected to lentiviral or retroviralintroduction (or other means of nucleic acid introduction) of thetransgene that encodes the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR, expandedto target therapeutic cell concentrations and infused into the patient,resulting in an autologous treatment with little graft vs hostcomplications. Although CARs have been shown to be very effective atachieving and sustaining remissions for refractory/relapsed acutelymphoblastic leukemia (Maude et al., NEJM, 371:1507, 2014, which isincorporated by reference in its entirety for all purposes), dangerousside effects related to cytokine release syndrome (CRS), tumor lysissyndrome (TLS), B cell aplasia or “on-tumor, off-target” toxicitiesoccur. Modulating CAR expression via the incorporation of DEs, RDEs, RNAcontrol devices, and/or Side-CARs in the Smart CAR(s), DE-CAR(s),RDE-CAR(s), Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s),Smart-DE-RDE-CAR(s) and/or Side-CAR(s) can control these toxicities.Modulating CAR expression via the incorporation of DEs, RDEs, RNAcontrol devices, and/or Side-CARs in the Smart CAR(s), DE-CAR(s),RDE-CAR(s), Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s),Smart-DE-RDE-CAR(s) and/or Side-CAR(s) can also minimize exhaustion,dysfunction and/or inhibition of the T-lymphocyte (or natural killercell) while providing a desired level of effector function.

T-cells can comprise CARs, DE-CARs, RDE-CARs and/or Side-CARs withintegrated RNA control devices. Combinational Smart DE-CAR,Smart-RDE-CAR, and/or Smart Side-CAR T-cells can be used, whereinindependent T-cells express orthogonal Smart CAR(s), DE-CAR(s),RDE-CAR(s), Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s),Smart-DE-RDE-CAR(s) and/or Side-CAR(s), that target distincttumor-associated antigens (TAAs). Targeting multiple TAAs simultaneouslycan direct a greater CTL response against the primary tumor ormetastases and prevent relapse. A potential disadvantage of using CAR,DE-CAR and/or Side-CAR polypeptide T-cells is that there may be a higherprobability of eliciting on-target, off-tumor effects, leading totoxicity. The coupling of DEs, RDEs, RNA control devices, and/orSide-CARs to CARs in Smart CAR(s), DE-CAR(s), RDE-CAR(s),Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s)and/or Side-CAR(s) T-cells mitigates the toxicity concern while enablingthe stronger response and relapse prevention of Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CAR T-cells. Smart-DE-CAR and/or Side-CAR T-cells can becontrolled by multiple ligands. The DE, RDE, RNA control devices, andSide-CARs can be used in combinational Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR T-cells are specific for different ligands, or combinations ofligands, such that expression cross-talk is minimized or eliminated. TheSmart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR T-cells can be used against a singletumor by targeting different tumor-associated surface antigens. TheseSmart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR T-cells can be used against a singletumor by targeting the same tumor-associated surface antigen, withdifferent transmembrane, hinge, receptor, costimulatory elements, otheraspects of the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR or combinations thereof.The Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR T-cells can be used against clonallyheterogeneous tumor types, wherein each population of Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CAR T-cell is specific for a particular TAA. The relativepopulations of Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR T-cells can be controlled.Combinations of ligands can be dosed to induce expression of a specificpopulation of Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR T-cells. Universal SmartCAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR T-cells can be used. Such CAR T-cellsare single T-cells that comprise more than one Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CAR or more than one means for Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR expression.

Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR and/or universal-CARs can be designed toinclude receptors against antigens that are of bacterial, fungal orviral origin. Because Smart CAR(s), DE-CAR(s), RDE-CAR(s),Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s)and/or Side-CAR(s) can be utilized to fight infections, which are asource of toxicity in immunocompromised patients, such anti-pathogenSmart CAR(s), DE-CAR(s), RDE-CAR(s), Smart-RDE-CAR(s), DE-RDE-CAR(s),Smart-DE-CAR(s), Smart-DE-RDE-CAR(s) and/or Side-CAR(s) can be used inconjunction Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR T-cell therapy specificfor a TAA.

A eukaryotic cell can bind to a specific antigen via the CAR, DE-CAR,and/or Side-CAR polypeptide causing the CAR, DE-CAR, and/or Side-CARpolypeptide to transmit a signal into the eukaryotic cell, and as aresult, the eukaryotic cell can be activated and so express anappropriate RDE-transgene. The activation of the eukaryotic cellexpressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR is varied depending on thekind of a eukaryotic cell and the intracellular element of the SmartCAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR. The eukaryotic cell can express a RDEtranscript that poises the cell for effector function upon stimulationof the eukaryotic cell through a Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR.

A eukaryotic cell expressing the RDE-transgene or RDE transcript, andoptionally, a Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, T-cell receptor, B-cellreceptor, innate immunity receptor and/or other receptor or targetingpolypeptide can be used as a therapeutic agent to treat a disease. Thetherapeutic agent can comprise the eukaryotic cell expressing theRDE-transgene or RDE transcript, and optionally, a Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, T-cell receptor, B-cell receptor, innate immunity receptorand/or other receptor or targeting polypeptide as an active ingredient,and may further comprise a suitable excipient. Examples of the excipientinclude pharmaceutically acceptable excipients for the composition. Thedisease against which the eukaryotic cell expressing the RDE-transgeneor RDE transcript, and optionally, a Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,T-cell receptor, B-cell receptor, innate immunity receptor and/or otherreceptor or targeting polypeptide is administered is not particularlylimited as long as the disease shows sensitivity to the eukaryotic celland/or the product of the RDE-transgene.

Examples of diseases that can be treated include a cancer (blood cancer(leukemia), solid tumor (ovarian cancer) etc.), an inflammatorydisease/autoimmune disease (asthma, eczema), hepatitis, and aninfectious disease, the cause of which is a virus such as influenza andHIV, a bacterium, or a fungus, for example, tuberculosis, MRSA, VRE, anddeep mycosis, other immune mediated diseases such as neurodegenerativediseases like Alzheimer's or Parkinson's, and metabolic diseases likediabetes. A receptor (e.g., a CAR) can target the eukaryotic cell to thediseased cell(s) and when the receptor binds to its target at thediseased cell(s) the receptor can send a signal into the eukaryotic cellleading to expression of the RDE-transgene. The RDE-transgene encodes apolypeptide that is useful in treating or killing the diseased cell(s).A cancer and/or solid tumor can be treated with a eukaryotic cellexpressing receptor that binds to a tumor associated (or cancerassociated) antigen, such as those described above. When the receptorbinds to the tumor associated antigen the receptor sends a signal intothe cell that causes the RDE-transgene to be expressed (e.g., the signaleffects an RDE binding protein leading to expression of theRDE-transcript). The RDE-transcript can encode a polypeptide thatactivates the eukaryotic cell so that the eukaryotic cell treats thecancer and/or the RDE-transcript encodes a polypeptide that itselftreats the cancer (e.g., a cytotoxic polypeptide).

An autoimmune disease (e.g., pemphigus vulgaris, lupus erythematosus,rheumatoid arthritis, multiple sclerosis, Crohn's disease) can betreated with a eukaryotic cell expressing a RDE-transgene or RDEtranscript, and optionally, a Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, T-cell receptor,B-cell receptor, innate immunity receptor and/or other receptor ortargeting polypeptide that binds to the immune proteins associated withthe autoimmune disease. The receptor or targeting polypeptide cantrigger expression of the RDE-transgene that encodes a polypeptideuseful in treating the autoimmune disease (e.g., the polypeptide canregulate the cells causing the autoimmune disease or kill these cells).The eukaryotic cell expressing the RDE-transgene or RDE transcript, andreceptor or targeting polypeptide can target cells that make an antibodyinvolved with the autoimmune disease (e.g., the RDE-transgene can encodea polypeptide that kills the antibody producing cells or that inhibitsthe production of antibody by these cells). The eukaryotic cellexpressing the RDE-transgene or RDE transcript, and receptor ortargeting polypeptide can target T-lymphocytes involved with theautoimmune disease (e.g., the RDE-transgene can encode a polypeptidethat kills the target T-lymphocytes or that regulates the activity ofthe T-lymphocytes).

Eukaryotic cells expressing the RDE-transgene or RDE transcript, andoptionally, a Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, T-cell receptor, B-cellreceptor, innate immunity receptor and/or other receptor or targetingpolypeptide can be used as a therapeutic agent to treat an allergy.Examples of allergies that can be treated include, for example,allergies to pollen, animal dander, peanuts, other nuts, milk products,gluten, eggs, seafood, shellfish, and soy. The eukaryotic cellexpressing the RDE-transgene or RDE transcript, and receptor ortargeting polypeptide can target cells that make an antibody whichcauses the allergic reaction against, for example, pollen, animaldander, peanuts, other nuts, milk products, gluten, eggs, seafood,shellfish, and soy. The targeted cells can be one or more of B-cells,memory B-cells, plasma cells, pre-B-cells, and progenitor B-cells.Targeted cells can also include T-lymphocytes which cause the allergicreaction against, for example, pollen, animal dander, peanuts, othernuts, milk products, gluten, eggs, seafood, shellfish, and soy.Eukaryotic cells expressing the RDE-transgene or RDE transcript, andreceptor or targeting polypeptide can bind to the idiotypic determinantof the antibody or T-cell receptor.

The eukaryotic cell expressing the RDE-transgene or RDE transcript, andoptionally, a Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, T-cell receptor, B-cellreceptor, innate immunity receptor and/or other receptor or targetingpolypeptide can be administered for treatment of a disease or condition.For example, the eukaryotic cell can be utilized to treat an infectiousdisease. The eukaryotic cell can express a receptor or targetingpolypeptide that binds to an antigen found on the infectious diseasecausing agent or a cell infected with such an agent. The receptor ortargeting polypeptide binds the antigen associated with the infectiousdisease and sends a signal into the eukaryotic cell that leads toexpression of the RDE-transgene. The RDE-transgene encodes a productthat can activate the eukaryotic cell for treating the infectiousdisease (e.g., the eukaryotic cell can produce a cytotoxic polypeptideor a cytokine that activates immune cells). The RDE-transgene can alsoencode a polypeptide that itself is a cytotoxic polypeptide or acytokine. The eukaryotic cell can also be utilized for prevention of aninfectious disease (used prophylactically), for example, after bonemarrow transplantation or exposure to radiation, donor lymphocytetransfusion for the purpose of remission of recurrent leukemia, and thelike.

The therapeutic agent comprising the eukaryotic cell expressing theRDE-transgene or RDE transcript, and optionally, a Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, T-cell receptor, B-cell receptor, innate immunity receptorand/or other receptor or targeting polypeptide as an active ingredientcan be administered intradermally, intramuscularly, subcutaneously,intraperitoneally, intranasally, intraarterially, intravenously,intratumorally, or into an afferent lymph vessel, by parenteraladministration, for example, by injection or infusion, although theadministration route is not limited.

The RDE-transgene or RDE transcript, and optionally, Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, T-cell receptor, B-cell receptor, innate immunity receptorand/or other receptor or targeting polypeptide can be used with aT-lymphocyte that has aggressive anti-tumor properties, such as thosedescribed in Pegram et al, CD28z CARs and armored CARs, 2014, Cancer J.20(2):127-133, which is incorporated by reference in its entirety forall purposes. The RDE transcript can encode a polypeptide that causesaggressive anti-tumor properties in the T-lymphocyte.

A transgene, a CAR, DE-CAR, and/or Side CAR polypeptides can becontrolled by an RDE from the 3′-UTR of the gene encoding IL-2 or the3′-UTR of IFN-γ. These RDEs can be modified to inactivate microRNA sitesfound in the RDE. Using these control elements makes expression of theCAR, DE-CAR, Side-CAR, and/or transgene sensitive to changes in theglycolytic state of the host cell through the interaction of the RDEwith glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When the hostcell is in a quiescent state a large proportion of the GAPDH is notinvolved in glycolysis and is able to bind to the RDE resulting inreduced translation of the transcript encoding the CAR, DE-CAR,Side-CAR, and/or transgene polypeptides. When the host cell is inducedto increase glycolysis, e.g., by providing the host cells with glucose,or other small molecules that will increase glycolytic activity, GAPDHbecomes enzymatically active and is not able to bind to the RDE. Thereduction in GAPDH binding to the RDE increases translation of thetranscripts (e.g., by increasing half-life of the transcript and/or byincreasing the translation rate) encoding the CAR, DE-CAR, Side-CAR, orother transgene. The glycolytic activity of GAPDH can be increased byincreasing the amount and/or activity of triose isomerase. The host cellcan be induced to over-express a recombinant triose isomerase, and thisover-expression increases the glycolytic activity of GAPDH. A glycolysisinhibitor can be added to decrease expression of the transcript with theRDE. Such glycolysis inhibitors include for example, rapamycin,2-deoxyglucose, 3-bromopyruvic acid, iodoacetate, fluoride, oxamate,pioglitazone, dichloroacetic acid, quinones, or other metabolisminhibitors such as, for example, dehydroepiandrosterone. Expression fromthe RDE controlled transcript can be increased by the addition of GAPDH(or other RDE binding protein) inhibitor that inhibits binding of theRDE by GAPDH (or other RDE binding protein). Such GAPDH inhibitorsinclude, for example, CGP 3466B maleate or Heptelidic acid (both sold bySanta Cruz Biotechnology, Inc.), pentalenolactone, or 3-bromopyruvicacid.

Constructs encoding transcripts with RDEs can be expressed in eukaryoticcells to bind to RDE binding proteins and so reduce the ability of thoseRDE binding proteins to interact with native transcripts in the cell.The recombinant transcripts can compete for binding of RDE bindingproteins and this can reduce the inhibition and/or activation of nativetranscripts within the eukaryotic cell by the RDE binding proteins. Theconstructs encoding transcripts with the RDEs can be used in this way tochange when and how native transcripts are expressed in the eukaryoticcell. The eukaryotic cell can be a T-cell, natural killer cell, orB-cell and the recombinant transcript has RDEs that are shared withcytokine or cytotoxic transcripts (e.g., in their 3′ untranslatedregions). The recombinant transcript can compete for binding with theRDE binding proteins (e.g., GAPDH and/or other glycolytic enzymesdescribed above) that regulate expression of the cytokine or cytotoxicpolypeptide and change the threshold (e.g., glycolysis activity forGAPDH) needed to express the cytokine or cytotoxic polypeptide. This canbe used to create super T-cell (aka Angry T-cells or Hornet T-cells)that will secrete higher amounts of cytokines and/or cytotoxic proteins(greater C_(max)) in response to stimulation of the immune cell (e.g.,through a CAR or T-cell receptor). T-cells can be reprogrammed with arecombinant transcript encoding an RDE from an IL-2 transcript so thatwhen the T-cell is stimulated by its T-cell receptor it produces moreIL-2 and other effector polypeptides with faster kinetics. Thesereprogrammed T-cells can also produce other inflammatory cytokines andcytotoxic polypeptides (e.g., granzymes and/or perforins) in largeramounts and with faster kinetics. Reprogramming T-cells and naturalkiller cells into such Angry/Hornet states can be useful for treatingdisease and disorders, including, for example, tumors, other cancers,and infectious diseases.

A subject can be diagnosed for certain diseases by identifying changesin the subject's RDEs. Such changes can cause gain or loss of functionto the RDE. For example, deletions, chromosomal rearrangements, andcertain base substitutions can cause the RDE to lose function (e.g., RNAbinding proteins that normally interact with the RDE no longer bind).Such loss of function changes can alter expression of the transcript andresult in aberrant expression. Detection of aberrant RDEs can be doneusing next generation nucleic acid sequencing, protein-RNA bindingassays or RNA binding protein trap methods such as those described inCastello et al., Molc. Cell 63:696-710 (2016), which is incorporated byreference in its entirety for all purposes. These RNA binding assays canbe used to diagnose change in function of a subject's RDEs. Thisaberrant expression can result in disease states such as cancerincluding, for example, adult T-cell leukemia/lymphoma, diffuse largeB-cell lymphoma, and stomach adenocarcinoma (these cancers have variantsin which the RDEs of the 3′-UTR for PD-L1 is altered leading to overexpression of PD-L1) (Kataoka K et al, Nature (2016) 534:402,doi:10.1038/nature18294, which is incorporated by reference in itsentirety for all purposes); inflammatory disease; autoimmune disease,such as, for example, systemic lupus erythematosus, type I diabetes,celiac disease (de Jong et al., Genes and Immunity (2016) 17, 75-78,which is incorporated by reference in its entirety for all purposes)(changes in the 3′-UTR of CTLA-4 that increased the length of the RDEcorrelated with autoimmunity), and rheumatoid arthritis (Tsuzaka et al.Modern Rheumatol. (2002) 12:167-173, which is incorporated by referencein its entirety for all purposes) (alternative splicing changed the RDEfor T-cell receptor ζ chain and resulted in down regulation ofexpression); and neurological disorders and pain disorders (Foulkes andWood, PLoS Genetics (2008) 4(7):e1000086, which is incorporated byreference in its entirety for all purposes).

Aberrant RDEs of a subject can be detected using sequencingtechnologies, amplification methods, hybridization based technologies,and other methods for detecting sequence changes. The RDEs can bedetected by analysis of the subject's nucleic acids (mRNA and/or DNA).Sequencing technologies include, for example, sanger sequencing, otherchain termination sequencing methods, Maxam and Gilbert sequencing,Polony sequencing, SOLID sequencing (sequencing by ligation), singlemolecule sequencing (e.g., Pacific Biosciences), ion torrent sequencing,pyrosequencing (e.g., 454 Life Sciences), sequencing by synthesis(Illumina), nanopore sequencing, etc. Amplification methods include, forexample, polymerase chain reaction (PCR), real-time PCR, transcriptionmediated amplification technologies, reverse transcriptase PCR, ligasechain reaction, strand displacement amplification, cleavase invader,etc. Hybridization methods include, for example, Southerns, Northerns,DNA-DNA hybridization, DNA-RNA hybridization, RNA-RNA hybridization,fluorescent in situ hybridization, etc. In other aspects, changes in theinteraction of RDEs with RDE binding proteins are identified usingprotein-RNA binding assays or RNA binding protein trap methods such asthose described in Castello et al., Molc. Cell 63:696-710 (2016), whichis incorporated by reference in its entirety for all purposes.

The inventions disclosed herein will be better understood from theexperimental details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the inventions as described more fully in theclaims which follow thereafter. Unless otherwise indicated, thedisclosure is not limited to specific procedures, materials, or thelike, as such may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

EXAMPLES Example 1. Control of T-Cell Effector Activity with a Smart-CAR

A Smart Car is made using the third generation anti-CD20 CAR cassettedescribed in Budde 2013 (Budde et al. PLoS1, 2013doi:10.1371/journal.pone.0082742, which is herebyincorporated-by-reference in its entirety for all purposes), and the RNAcontrol device, 3XL2bulge9 (Win and Smolke 2007 Proc. Natl Acad. Sci.104 (36): 14283-88, which is hereby incorporated by reference in itsentirety for all purposes). A nucleic acid encoding the 3XL2bulge9control device is engineered into the anti-CD20 CAR cassette in anappropriate expression vector.

This anti-CD20 Smart CAR is transfected by routine methods into T-cells(Jurkat cells and/or primary human T-cells), and stable populations ofT-cells are selected using appropriate antibiotics (or other selectionschemes). T-cell populations with anti-CD20 Smart CARs(CD20⁻/CD22⁻/CD3⁺) are activated by co-incubation with anti-CD3/CD28beads.

Activated anti-CD20 Smart CAR T-cells are co-cultured withCD20⁺/CD22⁺/CD3⁻ Ramos target cells at Smart CAR T-cell:Ramos targetratios of 2:1, 5:1, and 10:1. Ligand for the RNA control device,theophylline is added to the culture medium at concentrations in therange of 500 μM to 1 mM (lower or greater concentrations can be used totitrate Smart-CAR activity to the desired level). The Smart-CAR T-cellsand the Ramos cells are grown together for 48 hours. Cultures arewashed, and then stained with anti-CD22 and anti-CD3 reagents, followedby counting of CD22⁺ (Ramos target cells) and CD3⁺ cells (Smart CART-cells). These measurements will identify the target cell killing rate(e.g., half-life) and the proliferation rate of the Smart-CAR T-cells atdifferent levels of Smart-CAR expression.

Example 2. Control of T-Cell Effector Activity with CombinationSmart-CARs in a Human Subject

Nucleic acids encoding orthogonal Smart CARs that have specificity fordistinct TAAs and respond to distinct small molecule ligands areconstructed and are packaged into lentiviral vectors. Each of theseSmart CARs demonstrate in vitro cytotoxic T-cell effector function andantigen-dependent expansion in response to cognate ligand exposure, andindividually have known therapeutic windows in human patients.

To treat a human subject with tumors that express the defined set ofmultiple TAAs that are recognized by this Smart CAR pool, autologousT-cells are harvested from a patient's peripheral blood by apheresis andtransduced ex vivo with lentivirus encoding the cognate Smart CARs,either individually or in pools. Expanded Smart CAR CD4+ and/or CD8+T-cells are then adoptively transferred back into the patient. EachSmart CAR is individually activated with its own cognate small moleculeligand to initiate tumor recognition and elimination. As each Smart CARis individually controlled, therapeutic windows for each Smart CAR areadjusted to enforce maximal graft vs. tumor response, with tolerablegraft vs. host response. If the escape phase of tumor immunoediting isreached, the Smart CAR targeting the lost TAA is inactivated by removalof its cognate ligand to limit further graft vs. host response for aSmart CAR that no longer provides graft vs. tumor benefits. Bycontrolling Smart CAR toxicity and parallelizing a distributed attack onTAAs quickly, durable remissions for any tumor type are achieved.

Example 3. Control of T-Cell Effector Activity with a DE-CAR

A DE-CAR is made using the anti-CD20 CAR cassette described in Budde2013 (Budde et al. PLoS1, 2013 doi:10.1371/journal.pone.0082742, whichis hereby incorporated-by-reference in its entirety for all purposes),and the destabilizing element (DE) ecDHFR described in Iwamoto 2010(Iwamoto et al. Chemistry and Biology, 2010doi:10.1016/j.chembiol.2010.07.009, which is hereby incorporated byreference in its entirety for all purposes). The DE-CAR also can encodethe RNA control device, 3XL2bulge9 (Win and Smolke 2007 Proc. Natl Acad.Sci. 104 (36): 14283-88, which is hereby incorporated by reference inits entirety for all purposes). A nucleic acid encoding the DE of mutantscDHFR is engineered into the anti-CD20 CAR cassette in an appropriateexpression vector. Alternatively, a nucleic acid encoding the 3XL2bulge9control device is further engineered into the DE-anti-CD20 CAR cassette.

This anti-CD20 DE-CAR is transfected by routine methods into T-cells(Jurkat cells and/or primary human T-cells), and stable populations ofT-cells are selected using appropriate antibiotics (or other selectionschemes). T-cell populations with anti-CD20 DE-CARs or anti-CD20Smart-DE-CARs (CD20⁻/CD22⁻/CD3⁺) are activated by co-incubation withanti-CD3/CD28 beads.

Activated anti-CD20 DE-CAR T-cells or anti-CD20 Smart-DE-CAR T-cells areco-cultured with CD20⁺/CD22⁺/CD3⁻ Ramos target cells at DE-CAR T-cell(or Smart-DE-CAR T-cell):Ramos target ratios of 2:1, 5:1, and 10:1.Ligand for the DE, trimethoprim, and/or ligand for the RNA controldevice, theophylline, is added to the culture medium at concentrationsin the range of 500 μM to 1 mM (lower or greater concentrations can beused to titrate Smart-CAR activity to the desired level). The DE-CART-cells or Smart-DE-CAR T-cells and the Ramos cells are grown togetherfor 48 hours. Cultures are washed, and then stained with anti-CD22 andanti-CD3 reagents, followed by counting of CD22⁺ (Ramos target cells)and CD3⁺ cells (DE-CAR and/or Smart-DE-CAR T-cells). These measurementswill identify the target cell killing rate (e.g., half-life) and theproliferation rate of the Smart-CAR T-cells at different levels ofSmart-CAR expression.

Example 4. Increasing T-Cell Effector Activity with a Smart-CAR

A Smart Car is made using the third generation anti-CD19 CAR cassettedescribed in WO 2012/079000, which is hereby incorporated-by-referencein its entirety for all purposes), and the RNA control device,3XL2bulge9 (Win and Smolke 2007 Proc. Natl Acad. Sci. 104 (36):14283-88, which is hereby incorporated by reference in its entirety forall purposes). A nucleic acid encoding the 3XL2bulge9 control device isengineered into the anti-CD19 CAR cassette in an appropriate expressionvector.

The anti-CD19 Smart CAR and anti-CD19 CAR constructs are transfected byroutine methods into different populations of T-cells (Jurkat cellsand/or primary human T-cells), and stable populations of T-cells areselected using appropriate antibiotics (or other selection schemes).T-cell populations with anti-CD19 Smart CARs (CD19⁻/CD22⁻/CD3⁺) andT-cell populations with anti-CD19 CARs (CD19⁻/CD22⁻/CD3⁺) are activatedby co-incubation with anti-CD3/CD28 beads.

Activated anti-CD19 Smart CAR T-cells are co-cultured withCD19⁻/CD22⁻/CD3⁻ Ramos target cells at Smart CAR T-cell:Raji targetratios of 2:1, 5:1, and 10:1. Ligand for the RNA control device,theophylline is added to the culture medium at concentrations in therange of 2 μM to 2 mM (2 μM, 10 μM, 20 μM, 100 μM, 200 μM, 1 mM, and 2mM). The Smart-CAR T-cells and the Raji cells are grown together for 48hours. Cultures are washed, and then stained with anti-CD22 and anti-CD3reagents, followed by counting of CD22⁺ (Raji target cells) and CD3⁺cells (Smart CAR T-cells). These measurements will identify the targetcell killing rate (e.g., half-life) and the proliferation rate of theSmart-CAR T-cells at different levels of Smart-CAR expression.

Activated anti-CD19 Smart CAR T-cells are co-cultured withCD19⁻/CD22⁻/CD3⁻ Ramos target cells at Smart CAR T-cell:Raji targetratios of 2:1, 5:1, and 10:1. Ligand for the RNA control device,theophylline is added to the culture medium at concentrations in therange of 2 μM to 2 mM (2 μM, 10 μM, 20 μM, 100 μM, 200 μM, 1 mM, and 2mM). The Smart-CAR T-cells and the Raji cells are grown together for 48hours. Samples from culture media are taken and tested for IL-2 byELISA.

As a control activated anti-CD19 CAR T-cells are co-cultured withCD19⁻/CD22⁻/CD3⁻ Raji target cells at CAR T-cell:Raji target ratios of2:1, 5:1, and 10:1. Ligand for the RNA control device, theophylline isadded to the culture medium at concentrations in the range of 2 μM to 2mM (2 μM, 10 μM, 20 μM, 100 μM, 200 μM, 1 mM, and 2 mM). The CAR T-cellsand the Ramos cells are grown together for 48 hours. Cultures arewashed, and then stained with anti-CD22 and anti-CD3 reagents, followedby counting of CD22⁺ (Raji target cells) and CD3⁺ cells (CAR T-cells).These measurements will identify the target cell killing rate (e.g.,half-life) and the proliferation rate of the CAR T-cells at differentlevels of CAR expression.

As a control, activated anti-CD19 CAR T-cells are co-cultured withCD19⁺/CD22⁺/CD3⁻ Raji target cells at CAR T-cell:Raji target ratios of2:1, 5:1, and 10:1. Ligand for the RNA control device, theophylline isadded to the culture medium at concentrations in the range of 2 μM to 2mM (2 μM, 10 μM, 20 μM, 100 μM, 200 μM, 1 mM, and 2 mM). The CAR T-cellsand the Raji cells are grown together for 48 hours. Samples from culturemedia are taken and tested for IL-2 by ELISA.

Example 5: Target Cell Killing by CD8+ T-Lymphocytes with a Smart-CAR

A Smart Car was made using the anti-CD19 CAR described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes, and the RNA control device, 3XL2bulge9 (Win and Smolke2007 Proc. Natl Acad. Sci. 104 (36): 14283-88, which is herebyincorporated by reference in its entirety for all purposes). A nucleicacid encoding the 3XL2bulge9 control device was engineered into theanti-CD19 CAR cassette in an expression vector.

The anti-CD19 Smart CAR and anti-CD19 CAR constructs are transfected byroutine methods into different populations of T-cells (Jurkat cellsand/or primary human T-cells), and stable populations of T-cells areselected using appropriate antibiotics (or other selection schemes).T-cell populations with anti-CD19 Smart CARs (CD20−/CD22−/CD3+) andT-cell populations with anti-CD19 CARs (CD20−/CD22−/CD3+) are activatedby co-incubation with anti-CD3/CD28 beads.

T-cells with anti-CD19 Smart CARs or anti-CD19 CARs were incubated withtheophylline at 0, 75 and 250 μM for 72 hours. Activated anti-CD19 SmartCAR T-cells or anti-CD19 CAR T-cells were co-cultured withCD19+/CD22+/CD3− Raji target cells at Smart CAR T-cell:Raji targetratios of 2:1, 5:1, and 10:1. Ligand for the RNA control device,theophylline is maintained in the culture medium at concentrations of 0μM, 75 μM, and 250 μM. The Smart-CAR T-cells or CAR T-cells and the Rajicells are grown together for 18 hours. Cultures are washed, and thenstained with anti-CD22 and anti-CD3 reagents, followed by counting ofCD22+ (Raji target cells) and CD3+ cells (Smart CAR T-cells).

The results of this experiment are depicted in FIG. 6 , which showstarget cell killing graphed against time with individual curves for eachtheophylline concentration and for T-lymphocytes with anti-CD19 SmartCARs or T-lymphocytes with anti-CD19 CARs. This figure demonstrates animprovement in target cell killing with the theophylline controlledSmart CARs over the constitutively expressed CAR. The improvement wasabout 2 fold at 250 μM theophylline, and both 75 μM and 250 μMtheophylline improved target cell killing over the constitutivelyexpressed CAR. Both theophylline concentrations also showed about a twofold increase in cell killing over T-lymphocytes with the anti-CD19Smart CAR grown without theophylline (these cells also killed targetcells showing some basal level of CAR expression).

Example 6: Reducing T-Lymphocyte Exhaustion Markers forCD8+T-Lymphocytes with CD-19 CARs

A Smart Car was made using the anti-CD19 CAR described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes, and the RNA control device, 3XL2bulge9 (Win and Smolke2007 Proc. Natl Acad. Sci. 104 (36): 14283-88, which is herebyincorporated by reference in its entirety for all purposes). A nucleicacid encoding the 3XL2bulge9 control device was engineered into theanti-CD19 CAR cassette in an expression vector.

The anti-CD19 Smart CAR and anti-CD19 CAR constructs are transfected byroutine methods into different populations of T-cells (Jurkat cellsand/or primary human T-cells), and stable populations of T-cells areselected using appropriate antibiotics (or other selection schemes).T-cell populations with anti-CD19 Smart CARs (CD19−/CD22−/CD3+) andT-cell populations with anti-CD19 CARs (CD19−/CD22−/CD3+) are activatedby co-incubation with anti-CD3/CD28 beads.

T-cells with anti-CD19 Smart CARs or anti-CD19 CARs are incubated withtheophylline at 0, 75 and 250 μM for 72 hours. Activated anti-CD19 SmartCAR T-cells or anti-CD19 CAR T-cells are co-cultured withCD19+/CD22+/CD3− Raji target cells at Smart CAR T-cell:Raji targetratios of 2:1, 5:1, and 10:1. Ligand for the RNA control device,theophylline is maintained in the culture medium at concentrations of 0μM, 75 μM, and 250 μM. The Smart-CAR T-cells or CAR T-cells and the Rajicells are grown together for 18 hours. Cultures are washed, and thenstained with anti-CD22 and anti-CD3 reagents, followed by counting ofCD22+ (Raji target cells) and CD3+ cells (Smart CAR T-cells). The cellsare also stained with anti-PD-1, anti-TIM3 and/or anti-LAG-3 reagents tomeasure these T-lymphocyte exhaustion markers by ELISA or cell sorting.

The CD-19 Smart CAR T-lymphocytes will have lower levels of theexhaustion markers compared to the constitutively expressed CD-19 CART-lymphocytes. This reduction in exhaustion markers may be seen evenprior to stimulation of the CD-19 CAR lymphocytes and CD-19 Smart CARlymphocytes by CD-19 on target Raji cells.

Example 7. Control of T-Cell Effector Activity with an RDE-CAR

A RDE Car is made using the third generation anti-CD19 CAR cassettedescribed in WO 2012/079000, which is hereby incorporated-by-referencein its entirety for all purposes), and the 3′-UTR of the gene encodingIL-2 (NCBI Reference Sequence Number: NM 000586.3), which is herebyincorporated by reference in its entirety for all purposes). A nucleicacid encoding the IL-2 3′-UTR is engineered into the anti-CD19 CARcassette in an appropriate expression vector. The IL-2, 3′-UTR sequenceused was:

(SEQ ID NO: 28) taattaagtgcttcccacttaaaacatatcaggccttctATTTATTTAaatATTTAaattttatATTTAttgttgaatgtatggtttgctacctattgtaactattattcttaatcttaaaactataaatatggatcttttatgattctttttgtaagccctaggggctctaaaatggtttcacttATTTAtcccaaaatATTTAttattatgttgaatgttaaatatagtatctatgtagattggttagtaaaactATTTAataaatttgataaatataaa

The anti-CD19 RDE CAR and anti-CD19 CAR constructs are transfected byroutine methods into different populations of T-cells (primary humanT-cells), and stable populations of T-cells are selected usingappropriate antibiotics (or other selection schemes). T-cell populationswith anti-CD19 RDE CARs (CD19⁻/CD22⁻/CD3⁺) and T-cell populations withanti-CD19 CARs (CD19⁻/CD22⁻/CD3⁺) are activated by co-incubation withanti-CD3/CD28 beads and allowed to return to quiescent state afterdebeading.

Quiescent anti-CD19 RDE CAR T-cells are co-cultured withCD19+/CD22+/CD3⁻ Raji target cells at RDE CAR T-cell:Raji target ratiosof 2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The RDE-CAR T-cells and theRaji cells are grown together for 24 hours. Cultures are washed, andthen stained with anti-CD22 and anti-CD3 reagents, followed by countingof CD22⁺ (Raji target cells) and CD3⁺ cells (Smart CAR T-cells). Thesemeasurements will identify the target cell killing rate (e.g.,half-life) and the proliferation rate of the RDE-CAR T-cells atdifferent levels of RDE-CAR expression.

Activated anti-CD19 RDE CAR T-cells are co-cultured withCD19+/CD22+/CD3⁻ Raji target cells at RDE CAR T-cell:Raji target ratiosof 2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2, mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The RDE-CAR T-cells and theRaji cells are grown together for 24 hours. Samples from culture mediaare taken and tested for IL-2 by ELISA.

As a control activated anti-CD19 CAR T-cells are co-cultured withCD19+/CD22+/CD3⁻ Raji target cells at CAR T-cell:Raji target ratios of2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The CAR T-cells and the Rajicells are grown together for 24 hours. Cultures are washed, and thenstained with anti-CD22 and anti-CD3 reagents, followed by counting ofCD22⁺ (Raji target cells) and CD3⁺ cells (CAR T-cells).

As a control, activated anti-CD19 CAR T-cells are co-cultured withCD19+/CD22+/CD3⁻ Raji target cells at CAR T-cell:Raji target ratios of2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The CAR T-cells and the Rajicells are grown together for 48 hours. Samples from culture media aretaken and tested for IL-2 by ELISA.

Example 8: Removal of MicroRNA Binding Sites from an RDE

The AU-rich element from the 3′-UTR of IL-2 has mir-181 and mir 186microRNA binding sites. Different combinations of the microRNA siteswere removed from the 3′-UTR of IL-2. When the MIR186 micro-RNA siteswere removed from the 3′-UTR of IL-2 the dynamic range of expressionfrom constructs with this UTR increased 50 fold. The modified IL-2,3′-UTR replaces CTT in the sequence with GAA and is shown below (the newGAA is underlined in the sequence):

(SEQ ID NO: 29) taattaagtgcttcccacttaaaacatatcaggccttctATTTATTTAaatATTTAaattttatATTTAttgttgaatgtatggtttgctacctattgtaactattattcttaatcttaaaactataaatatggatcttttatgattGAAtttgtaagccctaggggctctaaaatggtttcacttATTTAtcccaaaatATTTAttattatgttgaatgttaaatatagtatctatgtagattggttagtaaaactATTTAataaatttgataaatataaa

The AU-rich element from the 3′UTR of IFNg also has micro-RNA bindingsites characterized as mir-125. The sequence of the IFNg RDE is:

(SEQ ID NO: 30) tggttgtcctgcctgcaatatttgaattttaaatctaaatctATTTAttaatATTTAacattATTTAtatggggaatatatttttagactcatcaatcaaataagtATTTAtaatagcaacttttgtgtaatgaaaatgaatatctattaatatatgtattATTTAtaattcctatatcctgtgactgtctcacttaatcctttgttttctgactaattaggcaaggctatgtgattacaaggctttatctcaggggccaactaggcagccaacctaagcaagatcccatgggttgtgtgtttatttcacttgatgatacaatgaacacttataagtgaagtgatactatccagttactgccggtttgaaaatatgcctgcaatctgagccagtgctttaatggcatgtcagacagaacttgaatgtgtcaggtgaccctgatgaaaacatagcatctcaggagatttcatgcctggtgcttccaaatattgttgacaactgtgactgtacccaaatggaaagtaactcatttgttaaaattatcaatatctaatatatatgaataaagtgtaagttcacaacta

Different combinations of the micro-RNA sites were removed from the3′UTR of IFNg and tested for increased expression. When the mir125micro-RNA sites were removed from the 3′-UTR of IFN-γ the expressionrate from constructs with this UTR is increased.

Expression of GFP in T-cells, transfected with the RDE-GFP plus themicroRNA sites, is compared to expression of GFP in T-cells with theRDE-GFP in which the microRNA sites have been removed, followingactivation with CD3/CD28 beads for 24 hours. The removal of the microRNAsites increased expression of the GFP by a factor of between 2-5 after24 hours, relative to the cells with microRNA sites.

Example 9: Control of a Chimeric Antigen Receptor for AML

A CAR is made using the anti-CD20 CAR cassette described in Budde 2013(Budde et al. PLoS1, 2013 doi:10.1371/journal.pone.0082742, which ishereby incorporated-by-reference in its entirety for all purposes), withthe anti-CD133 mAb 293C3-SDIE is used for the extracellular element(Rothfelder et al., 2015,ash.confex.com/ash/2015/webprogram/Paper81121.html, which isincorporated by reference in its entirety for all purposes) replacingthe anti-CD20 extracellular domain. The nucleic acid encoding theanti-CD133 CAR also can include the 3′-UTR from IL-2 engineered toremove the MIR186 micro-RNA sites or the 3′-UTR of IFN-γ engineered toremove the MIR125 micro-RNA sites, as described in Example 11. Aconstruct encoding the anti-CD20 CAR cassette is engineered to replacethe anti-CD20 extracellular domain with the anti-CD133 element, and the3′-UTR of IL-2 engineered to remove the MIR186 microRNA sites or the3′-UTR of IFN-γ engineered to remove the MIR125 micro-RNA sites, is alsoengineered into the construct. The anti-CD133 CAR with or without the3′-UTRs are cloned into appropriate expression vectors.

This anti-CD133 CAR or one of the anti-CD133 RDE-CARs (3′UTR of IL-2 or3′-UTR of IFN-γ) are transfected by routine methods into T-lymphocytes(Jurkat cells and/or primary human T-lymphocytes), and stablepopulations of T-lymphocytes are selected using appropriate antibiotics(or other selection schemes). T-lymphocyte populations withanti-anti-CD133 CAR, or the anti-CD133 RDE CAR (CD20⁻/CD22⁻/CD3⁺) areactivated by co-incubation with anti-CD3/CD28 beads with the addition ofa glycolysis inducer (e.g., glucose).

Activated anti-CD133 CAR, or the anti-CD133 RDE CAR T-lymphocytes areco-cultured with CD133⁺/CD3⁻ AML target cells (e.g., U937, MV4-11,MOLM-14, HL-60 and/or KG1a) at anti-CD133 CAR, or the anti-CD133 RDE CART-lymphocyte:AML target ratios of 2:1, 5:1, and 10:1. The anti-CD133 CARor the anti-CD133 RDE CAR T-lymphocytes and the AML cells are growntogether for 48 hours. Cultures are washed, and then stained withanti-CD133 and anti-CD3 reagents, followed by counting of CD133⁺ (AMLtarget cells) and CD3⁺ cells (anti-CD133 CAR, or the anti-CD133 RDE CART-lymphocytes). These measurements will identify the target cell killingrate (e.g., half-life) and the proliferation rate of the anti-CD133 CARor the anti-CD133 RDE CAR T-lymphocytes at different levels of CARexpression.

Example 10. An Off-Switch for Controlling T-Cell Effector Activity

A Smart-ZAP 70 mutant can be made using the Y319F ZAP 70 mutantdescribed in Wang et al., Cold Spring Harb Perspect Biol 2:a002279(2010), which is incorporated by reference in its entirety for allpurposes, and a Tet RNA control device described in Example 9. A nucleicacid encoding the Tet-RNA control device is operably linked to a nucleicacid encoding the Y319F ZAP 70 mutant, and this construct is in anappropriate expression vector.

T-cells with the anti-CD19 RDE CAR and anti-CD19 CAR constructs ofExample 8 are used in this example. The RNA control device Y319F ZAP 70is transfected by routine methods into T-cells with the anti-CD19 RDECAR and anti-CD19 CAR constructs made following Example 8, and stablepopulations of T-cells are selected using appropriate antibiotics (orother selection schemes). T-cell populations with anti-CD19 RDE CARs(CD19⁻/CD22⁻/CD3⁺) and T-cell populations with anti-CD19 CARs(CD19⁻/CD22⁻/CD3⁺) are activated by co-incubation with anti-CD3/CD28beads.

Activated RNA control device Y319F ZAP 70 and anti-CD19 RDE CAR T-cellsare co-cultured with CD19⁺/CD22⁺/CD3⁻ Ramos target cells at RDE CART-cell:Raji target ratios of 2:1, 5:1, and 10:1. The glycolysisactivator glucose is added to the culture medium at concentrations inthe range of 1.0 mM to 10 mM (1 mM, 2, mM, 3 mM, 4 mM, 5 mM, 7.5 mM and10 mM). Tetracycline in the range of 2 μM to 2 mM (2 μM, 10 μM, 20 μM,100 μM, 200 μM, 1 mM, and 2 mM) is added or not added to cells (havecell cultures±Tetracycline). The RNA control device Y319F ZAP 70+RDE-CART-cells and the Raji cells are grown together for 48 hours. Cultures arewashed, and then stained with anti-CD22 and anti-CD3 reagents, followedby counting of CD22⁺ (Raji target cells) and CD3⁺ cells (Smart CART-cells). These measurements will identify the target cell killing rate(e.g., half-life) and the proliferation rate of the RNA control deviceY319F ZAP 70+RDE-CAR T-cells versus the RDE-CAR T-cells at differentlevels of Y319F ZAP 70 expression and different levels of RDE-XAPexpression.

Example 11: Payload Delivery to DLBCL Using an Anti-CD19 CAR T-Cell

The anti-CD19 Smart CAR T-lymphocytes and anti-CD19 CAR T-celllymphocytes of Example 6 are used in this example. These CART-lymphocytes are further engineered to include a construct encoding aPD-1 inhibitor under the control of the 3′-UTR of IL2 that has beenmodified by removal of the MIR186 sites. PD-1 inhibitors expressed bythe construct include, for example, Pembrolizumab (Keytruda®), Nivolumab(Opdivo®), Atezolizumab (Tecentriq®), BMS-936558, Lambrolizumab, orpolypeptides derived from these drugs. Other PD-1 inhibitors that may beexpressed by the construct include those disclosed in Herbst et al., JClin Oncol., 31:3000 (2013); Heery et al., J Clin Oncol., 32:5s, 3064(2014); Powles et al., J Clin Oncol, 32:5s, 5011(2014); Segal et al., JClin Oncol., 32:5s, 3002 (2014), or U.S. Pat. Nos. 8,735,553; 8,617,546;8,008,449; 8,741,295; 8,552,154; 8,354,509; 8,779,105; 7,563,869;8,287,856; 8,927,697; 8,088,905; 7,595,048; 8,168,179; 6,808,710;7,943,743; 8,246,955; and 8,217,149.

T-cell populations with anti-CD19 Smart CARs/PD-1 (CD19−/CD22−/CD3+) andT-cell populations with anti-CD19 CARs/PD-1 (CD19−/CD22−/CD3+) areactivated by co-incubation with anti-CD3/CD28 beads. T-cells withanti-CD19 Smart CARs/PD-1 inhibitor or anti-CD19 CARs/PD-1 inhibitorwere incubated with theophylline at 0, 75 and 250 μM for 72 hours.Activated anti-CD19 Smart CAR/PD-1 T-cells or anti-CD19 CAR/PD-1 T-cellswere co-cultured with CD19+/CD22+/CD3− Raji target cells at SmartCAR/PD-1 T-cell:Raji target ratios of 2:1, 5:1, and 10:1. Ligand for theRNA control device, theophylline is maintained in the culture medium atconcentrations of 0 μM, 75 μM, and 250 μM. The Smart-CAR/PD-1 T-cells orCAR/PD-1 T-cells and the Raji cells are grown together for 18 hours.Cultures are washed, and then stained with anti-CD22 and anti-CD3reagents, followed by counting of CD22+ (Raji target cells) and CD3+cells (Smart CAR T-cells). Samples from culture media are also taken at6, 12 and 18 hours, and tested for PD-1 inhibitor by ELISA.

Example 12: Payload Delivery to AML Using an Anti-CD133 CAR T-Cell

A CAR is made using the anti-CD20 CAR cassette described in Budde 2013(Budde et al. PLoS1, 2013 doi:10.1371/journal.pone.0082742, which ishereby incorporated-by-reference in its entirety for all purposes), withthe anti-CD133 mAb 293C3-SDIE is used for the extracellular element(Rothfelder et al., 2015,ash.confex.com/ash/2015/webprogram/Paper81121.html, which isincorporated by reference in its entirety for all purposes) replacingthe anti-CD20 extracellular domain. The anti-CD133 CAR also can encodethe RNA control device, 3XL2bulge9 (Win and Smolke 2007 Proc. Natl Acad.Sci. 104 (36): 14283-88, which is hereby incorporated by reference inits entirety for all purposes). A nucleic acid encoding the anti-CD20CAR cassette is engineered to replace the anti-CD20 extracellular domainwith the anti-CD133 element, and optionally the RNA control device isalso engineered into the cassette. The anti-CD133 CAR with or withoutthe RNA control device are cloned into appropriate expression vectors.

These anti-CD133 CAR and anti-CD133 Smart CAR constructs are transfectedby routine methods into T-lymphocytes (Jurkat cells and/or primary humanT-lymphocytes), and stable populations of T-lymphocytes are selectedusing appropriate antibiotics (or other selection schemes).

These CAR T-lymphocytes are further engineered to include a constructencoding a PD-1 inhibitor under the control of the RDE from the 3′-UTRof IL2 that has been modified by removal of a MIR186 site. PD-1inhibitors expressed by the construct include, for example,Pembrolizumab (Keytruda®), Nivolumab (Opdivo®), Atezolizumab(Tecentriq®), BMS-936558, Lambrolizumab, or polypeptides derived fromthese drugs. Other PD-1 inhibitors that may be expressed by theconstruct include those disclosed in Herbst et al., J Clin Oncol.,31:3000 (2013); Heery et al., J Clin Oncol., 32:5s, 3064 (2014); Powleset al., J Clin Oncol, 32:5s, 5011(2014); Segal et al., J Clin Oncol.,32:5s, 3002 (2014), or U.S. Pat. Nos. 8,735,553; 8,617,546; 8,008,449;8,741,295; 8,552,154; 8,354,509; 8,779,105; 7,563,869; 8,287,856;8,927,697; 8,088,905; 7,595,048; 8,168,179; 6,808,710; 7,943,743;8,246,955; and 8,217,149.

T-lymphocyte populations with anti-CD133 CAR/PD-1 inhibitor oranti-CD133 Smart CAR/PD-1 inhibitor (CD20⁻/CD22⁻/CD3⁺) are activated byco-incubation with anti-CD3/CD28 beads.

Activated anti-CD133 CAR/PD-1 inhibitor or anti-CD133 Smart CAR/PD-1inhibitor T-lymphocytes are co-cultured with CD133⁺/CD3⁻ AML targetcells (e.g., U937, MV4-11, MOLM-14, HL-60 and/or KG1a) at anti-CD133 CARand/or anti-CD133 Smart CAR T-lymphocyte:AML target ratios of 2:1, 5:1,and 10:1. Ligand for the RNA control device, theophylline, is added tothe culture medium at concentrations in the range of 500 μM to 1 mM(lower or greater concentrations can be used to titrate Smart-CARactivity to the desired level). The anti-CD133 CAR/PD-1 inhibitor and/oranti-CD133 Smart CAR/PD-1 inhibitor T-lymphocytes and the AML cells aregrown together for 48 hours. Cultures are washed, and then stained withanti-CD133 and anti-CD3 reagents, followed by counting of CD133⁺ (AMLtarget cells) and CD3⁺ cells (anti-CD133 CAR, anti-CD133 DE-CAR,anti-CD133 Smart CAR, and/or the anti-CD133 DE-Smart CAR T-lymphocytes).These measurements will identify the target cell killing rate (e.g.,half-life) and the proliferation rate of the anti-CD133 CAR/PD-1inhibitor and/or anti-CD133 Smart CAR/PD-1 inhibitor T-lymphocytes atdifferent levels of CAR expression. Samples from culture media are alsotaken at 12, 24, 26 and 48 hours, and tested for PD-1 inhibitor byELISA.

Example 13: Constructs for Expressing a CAR and a Second Transgene

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a GFP-RDE2 (3′-UTR from IL-2) insert. These twoinserts/cassettes were placed on different lenti virus constructs orplaced in the same bicistronic lenti virus construct. In the bicistronicconstruct, the inserts with the anti-CD19 CAR and the insert with theGFP-RDE are transcribed in opposite directions, and the control regionsfor each are located in between the two insert/cassettes. The controlregion for the GFP-RDE2 insert in the single construct lenti vector wasMND (a synthetic promoter that contains the U3 region of a modifiedMoMuLV LTR with myeloproliferative sarcoma virus enhancer) which isdescribed in Li et al., J. Neurosci. Methods 189:56-64 (2010), which isincorporated by reference in its entirety for all purposes. The controlregion for the GFP-RDE2 insert in the bicistronic lenti vector was MinPand the RDE used the endogenous 3′-UTR of IL-2. The control region ofthe anti-CD19 CAR cassette in both constructs was also MND. CD4+ T-cellswere either doubly transduced with the individual anti-CD19 CAR lenticonstruct and the GFP-RDE2 lenti construct, or were singly transducedwith the bicistronic construct with both the anti-CD19 CAR lenticonstruct and the GFP-RDE2 lenti construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cells oranti-CD3/anti-CD28 beads were incubated with the transduced T cells for24h. At 24 h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and GFP expression in the T cells.

The doubly transduced T-cells showed an increase in fluorescence whencultured with Raji target cells (activate CAR) of 19.5% to 42.5% (about2 fold), and increase in fluorescence when cultured with CD3/CD28 beads(activate TCR) of 19.5% to 34.9% (about 1.8 fold). The doublytransformed T-cells showed a change in activated cells in the populationwhen cultured with Raji cells of 0.9% to 56.9%, and when cultured withCD3/CD28 beads of 0.9% to 92.5%.

The singly transduced T-cells showed a change in activated cells in thepopulation when cultured with Raji cells of 3.6% to 5.8%, and whencultured with CD3/CD28 beads of 3.6% to 6.6%.

Example 14: An RDE Construct for Expressing a Second Transgene

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a GFP-RDE1 (3′-UTR from IFNg) insert. These twoinserts/cassettes were placed in the same lenti virus construct. Theanti-CD19 CAR cassette and the insert with the GFP-RDE are transcribedin opposite directions, and the control regions for each are located inbetween the two insert/cassettes. The control region for the GFP-RDEinsert was MinP and the RDE was the endogenous 3′-UTR of IFNg. Thecontrol region of the anti-CD19 CAR cassette was MND (as describedabove). CD4+ T-cells were transduced with the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cells oranti-CD3/anti-CD28 beads were incubated with the transduced T cells for24h. At 24 h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and GFP expression in the T cells.

The transduced T-cells showed an increase in fluorescence when culturedwith Raji target cells (activate CAR) of 1.0% to 6.5% (about 6.5 fold),and increase in fluorescence when cultured with CD3/CD28 beads (activateTCR) of 1.0% to 4.4% (about 4.4 fold). The transformed T-cells showed achange in activated cells in the population when cultured with Rajicells of 0.9% to 84.8%, and when cultured with CD3/CD28 beads of 0.9% to90.8%.

Example 15: A Modified RDE2 Construct for Expressing a Second Transgene

Constructs were made using an anti-CD19 CAR cassette as described inExamples 11 and 12, and a GFP-RDE2.1 (IL-2 RDE) insert. The RDE2.1 wasmodified to remove the MIR186 microRNA sites, altering nucleotides fromthe 3′-UTR of IL-2 which was used as RDE2.

These two inserts/cassettes were placed in the same lenti virusconstruct. The anti-CD19 CAR cassette and the insert with the GFP-RDEare transcribed in opposite directions, and the control regions for eachare located in between the two insert/cassettes. The control region forthe GFP-RDE insert was a MinP. The control region of the anti-CD19 CARcassette in was MND (as described above). CD4+ T-cells were transducedwith the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cells oranti-CD3/anti-CD28 beads were incubated with the transduced T cells for24h. At 24 h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and GFP expression in the T cells.

The transduced T-cells showed a change in activated cells in thepopulation when cultured with Raji cells of 3.9% to 12.1%, and whencultured with CD3/CD28 beads of 3.9% to 11.1%.

Example 16: An RDE Construct for Expressing a Luciferase Transgene

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a Luciferase-RDE1 (3′-UTR of IFNg, Gold1) insertor a Luciferase-3′-UTR (a 3′-UTR that does not confer differentialtransgene translation in response to metabolic state of the cell,3′-UTR). The anti-CD19 CAR cassette and the insert with theluciferase-RDE1 are transcribed in opposite directions, and the controlregions for each are located in between the two insert/cassettes. Thecontrol region for the Luciferase-RDE1 insert and Luciferase-3′-UTR wereeither a MinP promoter or an NFAT promoter having the sequences of:

SEQ ID NO: 31 TAGAGGGTATATAATGGAAGCTCGACTTCCAG (MinP) SEQ ID NO: 32GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTAGATCTAGACTCTAGAGGGTATATAATGGAAGCTCGAATTC (NFAT)The control region of the anti-CD19 CAR cassette was the MND promoter.CD4+ T-cells were transduced with the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cells oranti-CD3/anti-CD28 beads were incubated with the transduced T cells for24h. At 24 h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and luciferase expression in the T cells.

FIG. 16 shows that the transduced T-cells had an increase inbioluminescence when cultured with Raji target cells (activate CAR) orwhen cultured with CD3/CD28 beads (activate TCR) as compared tobioluminescence of T-cells at resting. The T-cells with a NFAT promoterand the 3′-UTR of IFNg (Gold1) showed a larger on-off response from CARstimulation versus TCR stimulation. Under all conditions, T-cells withGold1 had lower amounts of bioluminescence than T-cells under the sameconditions (and same promoter) with Luciferase that was not controlledby the 3′UTR of IFNg (3′-UTR).

Example 17: Comparison of RDEs Controlling Luciferase

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a Luciferase-RDE1 (3′ UTR of IFNg, Gold1) insert,a Luciferase-RDE2 (3′-UTR of IL-2, Gold2) insert, a Luciferase-RDE3(3′-UTR of IL-2 modified as described above to remove the mir186 sites,Gold3), or a Luciferase-3′-UTR (a 3′-UTR that does not conferdifferential transgene translation in response to metabolic state of thecell, 3′-UTR). Combinations of these inserts/cassettes shown in FIG. 17were placed in the similar lenti virus constructs. The anti-CD19 CARcassette and the insert with the luciferase-RDE are transcribed inopposite directions, and the control regions for each are located inbetween the two insert/cassettes. The control region for theLuciferase-RDE insert and Luciferase-3′-UTR were either a MinP promoteror an NFAT promoter. The control region of the anti-CD19 CAR cassettewas the MND promoter, and CD4+ T-cells were transduced with thebicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set CD19+ Raji B cells were incubated with the transduced Tcells for 24h. At 24 h, the T cells were stained for CD25 and CD69,which are activation markers, and subject to flow cytometry to measurethese markers and luciferase expression in the T cells.

FIG. 17 shows that the transduced T-cells had an increase inbioluminescence when cultured with Raji target cells (activate CAR) ascompared to bioluminescence of T-cells at resting for constructs withRDE1 (Gold1), RDE2 (Gold2), or RDE3 (Gold3). The T-cells with a NFATpromoter and the RDE1 showed a larger on-off response than T-cells witha MinP promoter and the corresponding RDE. Under all conditions, T-cellswith an RDE controlling luciferase had lower amounts of bioluminescencethan T-cells with luciferase cassettes that were not controlled by anRDE. Combined with the MinP promoter, RDE1 gave a 4.1-fold increase inbioluminescence with CAR stimulation, RDE2 gave a 1.8-fold increase inbioluminescence, and RDE3 gave a 1.4-fold increase. Combined with theNFAT promoter, RDE1 gave a 8.5-fold increase in bioluminescence with CARstimulation, RDE2 gave a 3.1-fold increase in bioluminescence, and RDE3gave a 1.3-fold increase. With either promoter, the RDE3 construct gavethe highest amount of bioluminescence, the RDE1 construct gave thelowest amount of bioluminescence, and the RDE2 construct gave an amountof bioluminescence between RDE3 and RDE1.

Example 18: An RDE Construct for Expressing IL-12

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and an IL-12-RDE1 (3′-UTR of IFNg) insert or an IL-123′-UTR (a 3′-UTR that does not confer differential transgene translationin response to metabolic state of the cell). The anti-CD19 CAR cassetteand the insert with the IL-12-RDE1 are transcribed in oppositedirections, and the control regions for each are located in between thetwo insert/cassettes. The control region for the IL-12-RDE1 insert andIL-12 3′-UTR were either a minP promoter or an NFAT promoter. Thecontrol region of the anti-CD19 CAR cassette was the MND promoter. CD4+T-cells were transduced with the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set, CD19+ Raji B cells were incubated with the transduced Tcells for 24h. At 24 h, the T cells were stained for CD25 and CD69,which are activation markers, and subject to flow cytometry to measurethese markers. IL-12 expression in the T cells was measured by ELISA.

FIG. 18 shows that the transduced T-cells had an increase in IL-12expression when cultured with Raji target cells (activate CAR) ascompared to IL-12 expression of T-cells at resting using constructscontrolled by the MinP promoter or NFAT promoter. T-cells with the NFATpromoter and RDE1 (Gold1) showed a 168-fold change in IL-12 expressionform resting to CAR stimulation. T-cells with the NFAT promoter and a3′-UTR (not responsive to CAR stimulation, 3′-UTR) showed a 50-foldchange in expression, and a minP promoter with RDE1 (Gold1) showed a 6.3fold change in expression.

Disclosure of Python Code for Calculating Time Dependent EffectorFunction

import numpy as np

from math import exp

#E is effector activity, i.e. number of cytokines, perforins, etc. thatare produced

#nR=number of receptors

#nT=number of targets

#nRT=number of receptors and targets bound

#constants required for activation/decay calculations

c_act=1 #c_act=activation constant

c_inh=1 #c_inh=inhibition constant

c_eng=1 #c_eng=energy constant, a term available for metabolic function,currently not included

e_avail=1 #e_avail=energy available, a term available for metabolicfunction, currently not included

k1=1 #rxn constant for [r][t]→[rt]

k_1=1 #rxn constant for [rt]→[r][t]

k_car_to=0 #rate of car turnover

#constants required for ribozyme portion of model

k_rbz=1 #rxn constant for ribozyme

k_rbz_off=0.1 #rxn constant for ribozyme cleavage when in presence of

k_translation=1 #time/rate constant associated with translation of

def activation(y, t): #code for activation

E, nRT, nR, nT=y

dEdt=nRT*c_act−c_inh*E*t*nRT #t included in this term to simulateantigen dependent exhaustion maybe move to Qm

dnRTdt=k1*nR*nT−k_1*nRT

dnRdt=k_1*nRT−k1*nR*nT−nR*k_car_to

dnTdt=k_1*nRT−k1*nR*nT

return dEdt, dnRTdt, dnRdt, dnTdt

def decay(y, t): #code for natural decay via turnover

E, nRT, nR, nT=y

dEdt=nRT*c_act−c_inh*E*t*nRT

dnRTdt=k1*nR*nT−k_1*nRT

dnRdt=k_1*nRT−k1*nR*nT−nR*k_car_to

dnTdt=k_1*nRT−k1*nR*nT

return dEdt, dnRTdt, dnRdt, dnTdt

#t0=np.lnispace(0, 1, 11) #drug dosing from 0→1

t1=np.linspace(0, 1, 11) #activation from 1→2

t2=np.linspace(1, 5, 31) #decay from 1→5

from scipy.integrate import odeint

y1=[0.01, 0.01, 5, 10]

sol1=odeint(activation, y1, t1)

y2=[sol1[10,0],sol1[10,1],sol1[10,2], sol1[10,3]]

sol2=odeint(decay, y2, t2)

import matplotlib.pyplot as plt

plt.plot(t1, sol1[: 0], color=‘k’, marker=‘o’, label=‘E’)

plt.plot(t1, sol1[: 1], color=‘k’, marker=‘+’, label=‘nRT’)

plt.plot(t1, sol1[: 2], color=‘k’, marker=‘*’, label=‘nR’)

plt.plot(t1, sol1[: 3], color=‘k’, marker=‘s’, label=‘nT’)

plt.plot(t2, sol2[: 0], color=‘k’, marker=‘o’)

plt.plot(t2, sol2[: 1], color=‘k’, marker=‘+’)

plt.plot(t2, sol2[: 2], color=‘k’, marker=‘*’)

plt.plot(t2, sol2[: 3], color=‘k’, marker=‘s’)

pltlegend(loc=‘best’)

plt.xlabel(‘t’)

plt.grid( )

plt.show( )

All publications, patents and patent applications discussed and citedherein are incorporated herein by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A primary T-cell comprising: a chimeric antigenreceptor, and a heterologous nucleic acid comprising a promoter operablylinked to a polynucleotide encoding a transgene that is operably linkedto a polynucleotide encoding a RNA degradation element, wherein the RNAdegradation element is an AU rich element, wherein the heterologousnucleic acid is transcribed to make a transcript encoding the transgeneoperably linked to the RNA degradation element, and wherein the amountof polypeptide made from the transgene is increased after the chimericantigen receptor activates the primary T-cell and thereby changes ametabolic state of the primary T-cell.
 2. The primary T-cell of claim 1wherein the transgene encodes a cytokine, a FasL, an antibody, a growthfactor, a chemokine, an enzyme that cleaves a polypeptide or apolysaccharide, a granzyme, a perforin, or a checkpoint inhibitor. 3.The primary T-cell of claim 2, wherein the transgene encodes an IL-2, anIL-12, an IL-15, an IL-18, an IFNg, or a TNFα.
 4. The primary T-cell ofclaim 1, wherein the chimeric antigen receptor binds to a tumorassociated antigen.
 5. The primary T-cell of claim 4, wherein the tumorassociated antigen is a PSMA, a MUC1, a FAP, a folate receptor, aNY-ESO-1, or a CD33.
 6. The primary T-cell of claim 5, wherein thetransgene encodes a cytokine, a FasL, an antibody, a growth factor, achemokine, an enzyme that cleaves a polypeptide or a polysaccharide, agranzyme, a perforin, or a checkpoint inhibitor.
 7. The primary T-cellof claim 6, wherein the transgene encodes an IL-2, an IL-12, an IL-15,an IL-18, an IFNg, or a TNFα.
 8. The primary T-cell of claim 4, whereinthe tumor associated antigen is a MUC1, a FAP, or a folate receptor,wherein the tumor associated antigen is on a cancer cell.
 9. The primaryT-cell of claim 8, wherein the transgene encodes a cytokine, a FasL, anantibody, a growth factor, a chemokine, an enzyme that cleaves apolypeptide or a polysaccharide, a granzyme, a perform, or a checkpointinhibitor.
 10. The primary T-cell of claim 9, wherein the transgeneencodes an IL-2, an IL-12, an IL-15, an IL-18, an IFNg, or a TNFα. 11.The primary T-cell of claim 8, wherein the tumor associated antigen is aMUC1, and wherein the tumor associated antigen is on a pancreatic cancercell or a breast cancer cell.
 12. The primary T-cell of claim 11,wherein the transgene encodes a cytokine, a FasL, an antibody, a growthfactor, a chemokine, an enzyme that cleaves a polypeptide or apolysaccharide, a granzyme, a perform, or a checkpoint inhibitor. 13.The primary T-cell of claim 12, wherein the transgene encodes an IL-2,an IL-12, an IL-15, an IL-18, an IFNg, or a TNFα.
 14. The primary T-cellof claim 4, wherein the tumor associated antigen is a NY-ESO-1, andwherein the tumor associated antigen is on a melanoma cell.
 15. Theprimary T-cell of claim 14, wherein the transgene encodes a cytokine, aFasL, an antibody, a growth factor, a chemokine, an enzyme that cleavesa polypeptide or a polysaccharide, a granzyme, a perforin, or acheckpoint inhibitor.
 16. The primary T-cell of claim 15, wherein thetransgene encodes an IL-2, an IL-12, an IL-15, an IL-18, an IFNg, or aTNFα.
 17. The primary T-cell of claim 4, wherein the tumor associatedantigen is a CD33, and wherein the tumor associated antigen is on anAML, cell.
 18. The primary T-cell of claim 17, wherein the transgeneencodes a cytokine, a FasL, an antibody, a growth factor, a chemokine,an enzyme that cleaves a polypeptide or a polysaccharide, a granzyme, aperforin, or a checkpoint inhibitor.
 19. The primary T-cell of claim 18,wherein the transgene encodes an IL-2, an IL-12, an IL-15, an IL-18, anIFNg, or a TNFα.
 20. The primary T-cell of claim 13, wherein the RNAdegradation element is from a 3′-UTR of IFNg or a 3′-UTR of IL-6.